Bioreactor and methods of use thereof

ABSTRACT

An inverted conical bioreactor is provided for growing cells or microorganisms. The bioreactor has an internal space and a perforated barrier within the vessel, through which a liquid may flow, where cells or microorganisms cannot pass through the perforated barrier. The perforated barrier divides the internal space of the bioreactor into a first chamber and a second chamber. Cells are grown within the second chamber and can be perfused by re-circulating the liquid, for example a growth medium, through the bioreactor. Various inlet ports and outlet ports allow controlling the parameters of flow of the growth medium.

FIELD OF DISCLOSURE

Bioreactors comprising a perforated barrier for growing living cells ormicroorganisms are disclosed herein. Methods for growing cells ormicroorganisms in the bioreactors described herein, wherein regulationof flow-rates may be used for growth of cells or microorganisms atdifferent densities.

BACKGROUND

Bioreactors are used to culture microorganisms and isolated livingcells, including mammalian and human cells, in a contained andcontrolled environment. In many cases, the culturing of microorganismsand cells require the microorganisms or cells be physically separatedand isolated from the surrounding environment and maintained in asterile environment. Such cases can include the development andmanufacturing of therapeutic microorganisms or cells, such as vaccinesand genetically modified cells, and the manufacturing of tools fortherapy such as viruses for gene therapy, proteins, antibodies ortherapeutic cells. Additionally, the need for containment of themicroorganism or cell from the environment could be in cases in whichthe organism is hazardous.

Culturing and processing of such microorganisms and cells requiresseveral typical steps that might include, but are not limited to,inoculating a bioreactor with a small number of organisms or cells,constantly supplying the microorganism or cells with nutrients, media,supplements, activators, measuring microorganism or cell number,maintaining viability, maintaining identity of the microorganism orcell, maintaining the physical state, and cell collection. During growthand expansion of microorganisms and cells in a bioreactor, it is alsoimportant to monitor parameters such as media and glucose consumption,Oxyen, H+ ions in media, conductivity and more. Additionally, long termculturing will usually include transfer of the microorganisms or cellsto larger containers as they proliferate. Once the number ofmicroorganism or cells reaches the needed number or activity, themicroorganisms or cells are usually processed and formulated. Suchprocessing can include washing of the growth media, concentrating thecells or microorganisms, replacing the media to the final preservationmedia, or packaging and freezing the microorganisms or cells for furtheruse.

Bioreactors may be used for growing, proliferating, differentiating andmaintaining living cells and/or microorganisms for different purposes.Cells grown in such bioreactors are typically perfused by a growthmedium, which provides nutrients and oxygen to the cells and removeswaste materials and carbon dioxide excreted by the cells. Typically,various steps may be performed before and/or during the culturing ofcells or microorganisms in such bioreactors including, for example,selecting cells, culturing cells, modifying cells, activating the cells,expanding the cells (by cell proliferation), washing the cells,concentrating the cells and final formulating of the cells (ormicroorganisms).

To date, propagation is commonly performed by transferring the mediumwith the microorganisms or cells between different containers andvarious tools are used for this purpose, such as larger growth vessels,centrifugation tubes or bags, intermediate storage containers and thefinal packaging. The above processes may typically include openmanipulations were the microorganisms or cells are transferred from onestep to the other.

Several of the above indicated steps may require removing the cells fromthe bioreactor and further subjecting them to steps such as, amongothers centrifugation, separation, incubation, counting, testing,separation, formulation and packaging. Unfortunately, any stepsinvolving taking the cells or microorganisms out of the bioreactorsignificantly increase the risk of contamination of the cell by unwantedmicroorganisms (such as, for example, fungi, bacteria, mycoplasma orother undesired microorganisms) which may adversely compromise the cellculturing process.

There is a long felt need for closed system bioreactors that may reduceor eliminate the need to process the cells or microorganisms by takingthem out of the bioreactor and reduce or eliminate the steps and humaninteraction with the cells during the culture. Furthermore, there is aneed to automate and optimize the process end to end by processing thecells from early stages to a final product in one automated and closedsystem. The bioreactors described herein address these needs and furtherprovide advantageous growth conditions allowing for higher yields andlower media needs.

SUMMARY

In one aspect, disclosed herein is a bioreactor for growing cells ormicroorganisms therein, the bioreactor comprising:

-   -   a closed vessel enclosing a space therein;    -   a first barrier having a plurality of pores therein, the first        barrier is sealingly disposed within the space configured to        divide the space into a first chamber and a second chamber,        wherein the second chamber is configured to accommodate the        growing cells or microorganisms therein, and wherein a diameter        of the pores is configured to allow a fluid flow solely between        the first chamber and the second chamber and vice versa,    -   one or more fluid inlet ports for introducing the fluid into the        first chamber; and    -   one or more fluid outlet ports for allowing the fluid to exit        from the second chamber.

In some related aspects, the first barrier does not allow cells ormicroorganisms grown in the vessel to pass between the first chamber andthe second chamber.

In some related aspects, the first chamber is a lower chamber and thesecond chamber is an upper chamber and wherein the fluid flow comprisesan upstream flow

In some related aspects, the first barrier is disposed in contact withwalls of the vessel.

In some related aspects, the bioreactor further comprises an aligningbarrier having a plurality of pores therein; the aligning barrier issealingly disposed within the space of the first chamber under the firstbarrier; the aligning barrier is configured to align the fluid flow andprevent bubbles passage.

In some related aspects, the aligning barrier is configured to controlvelocity of the fluid flow.

In some related aspects, the pores of the aligning barrier compriseconical shapes.

In some related aspects, the bioreactor further comprises an additionalscreening barrier having a plurality of pores therein; the screeningbarrier is disposed within the space of the second chamber, at topsection of the second chamber, such that the growing cells ormicroorganisms are accommodated between the first barrier and thescreening barrier; the screening barrier is configured to prevent thecells passage.

In some related aspects, the bioreactor vessel is constructed of atleast two parts.

In some related aspects, the vessel of the bioreactor is configured toprovide a fluid velocity gradient in the fluid disposed within thesecond chamber, such that the velocity of the fluid decreases in adirection from the first barrier towards a top surface of the fluid.

In some related aspects, at least the second chamber comprises anincreasing transversal cross sectional area from bottom to top of thesecond chamber.

In some related aspects, the shape of the transversal cross sections isselected from: a circle, an ellipse, a polygon, and any combinationthereof.

In some related aspects, the shape of the vessel is selected from: aconical shape, a frustoconical shape, a tapering shape, a cylindricalshape, a polygonal prism shape, a tapering shape having an ellipsoidaltransversal cross section, a tapering shape having a polygonaltransversal cross section, a shape having a cylindrical part and atapering part and a shape having a conical or tapered part and ahemispherical part, and any combination thereof.

In some related aspects, at least one of the one or more fluid outletports is configured to be fluidically connected to a pump, which isconfigured to receive the fluid from the second chamber, and optionallywherein the pump is further configured to recirculate the fluid backinto the first chamber via a t least one of the fluid inlet ports.

In some related aspects, the rate of flow of the fluid through thesecond chamber is controlled by the pump's pumping rate.

In some related aspects, the fluid comprises any one of: a growth media,a washing solution, a nutrient solution, a collection solution, aharvesting solution, a storage solution, and any combination thereof.

In some related aspects, wherein the one or more fluid outlet portscomprise a plurality of fluid outlet ports opening at differentpositions along the height of the second chamber.

In some related aspects, the first barrier is a fixed non-movablebarrier.

In some related aspects, the fixed barrier is selected from: a flatbarrier, a flat barrier inclined at an angle to a longitudinal axis ofthe bioreactor, a concave barrier with a concave upper surface facingtop of the vessel, a tapering barrier and a conical barrier.

In some related aspects, the bioreactor further comprises at least oneharvesting port disposed in the vicinity of an upper surface of thefirst barrier configured to harvest cells from the bioreactor.

In some related aspects, the bioreactor is configured to be inverted.

In some related aspects, the bioreactor further comprises a supportingmatrix disposed within the second chamber for supporting the cells ormicroorganisms.

In some related aspects, the bioreactor further comprises a controlleris operably coupled and configured to control at least to one of:

at least one sensor unit comprising one or more sensors configured tosense one or more chemical and/or physical properties of the fluidwithin the vessel;

a plurality of controllably openable and closable valves configured tocontrol the flow the fluid within the one or more fluid outlet portsoutlet and fluid inlet ports;

-   -   a controllably openable and closable valve configured to control        the flow of fresh liquid fluid from a fluid reservoir into an        inlet port of the the pump;    -   a heater unit configured to heat the fluid within the vessel;    -   a cooling unit configured to cool the fluid within the vessel;        and    -   a gas valve configured to control the flow of a gas comprising        oxygen from an oxygen source into a gas dispersing head disposed        within the vessel.

In a related aspect, a method for growing cells or microorganisms isdisclosed, in a bioreactor of according to any one of the above aspects,the method comprises the steps of:

-   -   introducing cells or microorganisms into the second chamber of        the bioreactor;    -   perfusing the cells or microorganisms with the fluid;    -   growing the cells to a desired concentration; and    -   harvesting the cells or microorganisms from the bioreactor.

In some related aspects, the step of perfusing comprises controlling thelevel and/or the rate of flow of the fluid within the bioreactor.

In some related aspects, the step of perfusing comprises re-circulatingthe fluid through the first barrier.

In some related aspects, the step of re-circulating further comprises atleast one of:

-   -   a step of adding an amount of fresh fluid to the bioreactor; and    -   a step of draining an amount of the fluid from the bioreactor.

In some related aspects,

-   -   the step of perfusing further comprises a step of oxygenating        the fluid; or    -   the step of perfusing further comprises controlling the level        and/or the rate of flow of the fluid within bioreactor; or    -   the method further comprises step of increasing the level of the        fluid in the second chamber; or    -   the method further comprises one or more steps of washing the        cells or microorganisms; or    -   the method further comprises a step of concentrating the cells        by reducing the volume of the fluid within the second chamber;        or    -   the method further comprises a step of maintaining the cell mass        in a floating position at a specific region in the second        chamber, due to a balance between gravity force applied on the        cell mass and selected velocity of the upstream fluid flow; or    -   any combination thereof.

In some related aspects, the cells are adherent cells and the methodfurther comprises a step of allowing the cells to attach to one or moresurfaces disposed within the second chamber.

In some related aspects, the one or more surfaces are selected from thegroup consisting of, the upper surface of the first barrier, the surfaceof the walls of the second chamber, the surface of a cell supportingmatrix disposed within the second chamber and any combination thereof.

In some related aspects, the method further comprises a step ofco-culturing the cells with additional different cells.

In some related aspects,

-   -   the cells are T-cells and the additional different cells are        cytokine secreting cells; or    -   the cells are T-cells and the additional different cells are        antigen presenting cells; or    -   the cells are embryonic stem cells and the additional different        cells are feeder cells.

In some related aspects, the steps of introducing, perfusing, growing,washing and harvesting the cells are continuous and performed in or fromthe second chamber.

In one aspect, disclosed herein is a bioreactor for growing cells ormicroorganisms therein, the bioreactor comprising: a vessel having avessel wall enclosing a space therein; a perforated barrier having aplurality of perforations therein, the barrier is sealingly disposedwithin the space to divide the space into a first chamber and a secondchamber, wherein the diameter of the perforations is configured to allowsolely a liquid flow from the first chamber to the second chamber andfrom the second chamber to the first chamber, one or more fluid inletports for introducing the liquid into the first chamber; and one or morefluid outlet ports for allowing the liquid to exit the second chamber.

In a related aspect, the bioreactor further comprises a fluid impellerdisposed within the first chamber and fluidically coupled to the one ormore fluid inlet port. According to some embodiments, the fluid impellercomprises a hollow member having a plurality of perforations and/orfluid nozzles therein configured for ejecting multiple jets of a liquidwithin the first chamber when the liquid is pumped into the one or morefluid inlet port. In another related aspect, the bioreactor furthercomprises a gas dispersing head configured for providing oxygen to theliquid.

In another related aspect, one or more fluid outlet ports comprises asingle fluid outlet port, and the one or more inlet ports comprises asingle fluid inlet port, and wherein the fluid inlet port is configuredfor introducing the liquid into the first chamber by a pump fluidicallyconnected to the fluid inlet port, wherein the pump is configured tofluidically connect to the single fluid outlet port and configured toreceive the liquid from the second chamber, and configured forrecirculating the liquid within the bioreactor.

In a related aspect, the rate of flow of the liquid through the secondchamber is controlled by controlling the rate of pumping of the liquidby the pump. In another related aspect, the liquid comprises a growthmedia, a washing solution, a nutrient solution, a collection solution, aharvesting solution, a storage solution, or any combination thereof.

In a related aspect, one or more fluid inlet port comprises one fluidinlet port and the one or more fluid outlet ports comprise a pluralityof fluid outlet ports opening at different positions along the height ofthe second chamber, and wherein the plurality of fluid outlet ports areconfigured to each be fluidically connectable to a fluid manifold,wherein the fluid manifold is fluidically connected to a pump such thatany selected fluid output port of the plurality of fluid outlet ports isconfigured to be fluidically controllably connected to the pump by thefluid manifold for receiving the liquid from the second chamber into thepump through the selected fluid output port and for introducing theliquid by the pump into the first chamber through the single fluid inletport, wherein the level of the liquid within the second chamber isdetermined by the fluid outlet port selected from the plurality of fluidoutlet ports.

In another related aspect, the bioreactor further comprises a pluralityof valves, each fluid outlet port of the plurality of fluid outlet portsis configured to be fluidically coupled to a valve of the plurality ofvalves, and wherein the fluid manifold is configured to be fluidicallyselectably connectable to any selected fluid outlet port of theplurality of fluid outlet ports through the valve connected to the fluidoutput port. In another related aspect, the bioreactor further comprisesa temperature control unit configured for regulating the temperature ofthe liquid disposed within the bioreactor. In another related aspect,the temperature control unit is selected from: a heating element, acooling element, and a combination of a heating element and a coolingelement.

In a related aspect, the bioreactor is configured for establishing afluid velocity gradient in the liquid disposed within the second chambersuch that the velocity of the liquid in the second chamber graduallydecreases in the direction from the perforated bather towards the topsurface of the liquid in the second chamber. In another related aspect,the fluid velocity gradient in the liquid is achieved by the transversalcross sectional area of the top part of the second chamber being largerthan the transversal cross sectional area of the bottom part of thesecond chamber.

In another related aspect, the shape of transversal cross sections ofthe second chamber is selected from a circle, an ellipse, a polygon, anda regular polygon. In another related aspect, the vessel walls of thebioreactor comprise one or more closable and/or sealable openings formedtherein. In another related aspect, one or more closable and/or sealableopenings are selected from one or more openings disposed in the top partof the bioreactor, and one or more openings disposed in the side wallsof the bioreactor, and any combinations thereof.

In a related aspect, the bioreactor further comprises a self-sealinggasket sealingly disposed in the vessel walls and configured forinserting of a syringe needle through the gasket for injecting the cellsor microorganisms into the second chamber through the needle.

In a related aspect, the shape of the bioreactor is selected from aconical shape, a frustoconical shape, a tapering shape, a cylindricalshape, a polygonal prism shape, a tapering shape having an ellipsoidaltransversal cross section, a tapering shape having a polygonaltransversal cross section, a shape having a cylindrical part and atapering part and a shape having a conical or tapered part and ahemispherical part, or a combination thereof.

In a related aspect, the perforated barrier is a fixed non-movableperforated barrier. In another related aspect, the fixed perforatedbarrier is selected from, a flat perforated barrier, a flat perforatedbarrier inclined at an angle to a longitudinal axis of the bioreactor, aconcave perforated barrier with a concave upper surface facing the topof the bioreactor, a tapering perforated barrier and a conicalperforated barrier. In another related aspect, the perforated barrier isa movable perforated barrier. In another related aspect, the movableperforated barrier is selected from, a movable perforated barriersealingly attached to the vessel walls by a flexible and/or stretchablemember the flexible and/or stretchable member is sealingly attached to aperimeter of the perforated barrier and sealingly attached to the vesselwall, a deformable and/or flexible perforated barrier, and a convexbuckling perforated barrier with a convex upper surface facing the topof the bioreactor. In another related aspect, the perforated barrierfurther comprises a magnetic member attached thereto for enabling movingand/or tilting and/or deforming and/or buckling of the perforatedbarrier by applying force to the perforated barrier using a magnetdisposed outside of the bioreactor. In another related aspect, theperforated barrier does not allow cells or microorganisms grown in thevessel to pass through the perforated barrier from the first chamber tothe second chamber and the second chamber to the first chamber.

In a related aspect, the bioreactor further comprises an additionalperforated barrier within the first chamber between the bottom of thevessel and the perforated barrier that separates the first and secondchambers, or an additional perforated barrier within the second chamberbetween the cells and the top of the vessel, or a combination thereof.

In a related aspect, the bioreactor further comprises at least oneharvesting port disposed in the vessel walls and opening into the secondchamber in the vicinity of an upper surface of the perforated barrierconfigured for harvesting cells from the bioreactor. In a relatedaspect, the bioreactor further comprises a harvesting port including ahollow member having a first end sealingly attached to the perforatedbarrier and opening at an upper surface of the perforated barrier, and asecond end sealingly passing through the walls of the first chamber andcloseably opening outside the bioreactor. In another related aspect, thebioreactor includes at least one harvesting port disposed in the vesselwalls and opening into the second chamber in the vicinity of an uppersurface of the perforated barrier, and wherein the bioreactor is atiltable bioreactor configured to be tilted at an angle to a verticaldirection to assist the harvesting of cells through the at least oneharvesting port.

In a related aspect, the bioreactor is configured to be inverted.

In a related aspect, the bioreactor further comprises anopenable/closable outlet port disposed in the walls or bottom part ofthe first chamber configured for draining at least some of the liquidfrom the bioreactor. In another related aspect, the bioreactor isconfigured to be fluidically connected to a pump fluidically couplableto a fluid reservoir disposed outside of the bioreactor for introducingfresh liquid from the fluid reservoir into the bioreactor.

In a related aspect, the bioreactor further comprises at least onesensor unit comprising at least one sensor configured for sensing one ormore chemical and/or physical properties of the liquid.

In a related aspect, the bioreactor is operationally couplable to acontroller for controlling the operation of the bioreactor.

In a related aspect, the bioreactor further comprises a fluid impellerdisposed within the first chamber and fluidically coupled to at leastone fluid inlet port of the one or more fluid inlet ports, the fluidimpeller comprises a hollow member having a plurality of perforationsand/or fluid nozzles therein configured for ejecting multiple jets of aliquid within the first chamber when the liquid is pumped into the atleast one fluid inlet port. In another related aspect, the one or morefluid inlet ports and the one or more fluid outlet ports comprise or areconfigured to be fluidically connected to valves for controllablyopening and closing the one or more fluid inlet ports and the one ormore fluid outlet ports. In another related aspect, the valves areselected from manually operable valves and automatically operable valvesconnectable to a controller. In another related aspect, theautomatically operable valves are electrically actuated solenoid basedvalves connectable to a controller for automatically controlling theopening and closing of the valves.

In a related aspect, the bioreactor further comprises a supportingmatrix disposed within the second chamber for supporting the cells ormicroorganisms.

In one aspect, this application discloses a bioreactor systemcomprising: a bioreactor as disclosed herein; and a pump for circulatinga liquid within the bioreactor.

In a related aspect, the pump receives liquid from the one or more fluidoutlet ports and pumps the received liquid into the one or more fluidinlet ports. In another related aspect, the bioreactor system furthercomprises a fluid reservoir fluidically couplable to an inlet port ofthe pump for controllably providing fresh liquid to the pump to bepumped into the first chamber.

In a related aspect, the bioreactor system further comprises acontroller for manually or automatically controlling the operation ofthe bioreactor. In another related aspect, the controller is operablycoupled to one or more of, at least one sensor unit comprising one ormore sensors for sensing one or more chemical and/or physical propertiesof the liquid, a plurality of controllably openable and closable valvesfor controlling the flow of the liquid within the one or more fluidoutlet ports outlet, a controllably openable and closable valve forcontrolling the flow of fresh liquid from a fluid reservoir into aninlet port of the pump, a heater unit for heating the liquid, a coolingunit for cooling the liquid, and a gas valve for controlling the flow ofa gas comprising oxygen from an oxygen source into a gas dispersing headdisposed within the bioreactor.

In a related aspect, the bioreactor further comprises a supportingmatrix disposed within the second chamber for supporting the cells ormicroorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter disclosed herein is particularly pointed out anddistinctly claimed in the concluding portion of the specification.However, the bioreactors disclosed herein, both as to organization andmethod of operation, together with objects, features, and advantagesthereof, may best be understood by reference to the following detaileddescription when read with the accompanying drawings in which:

FIG. 1 is a schematic part cross-sectional view illustrating someembodiments of a bioreactor system disclosed herein, wherein the systemcomprises a bioreactor comprising a perforated barrier;

FIG. 2 is a schematic part cross-sectional view illustrating someembodiments of a bioreactor system disclosed herein comprising abioreactor with multiple fluid outlet ports for controllably adjustingthe level of the growth medium in the bioreactor;

FIG. 3 is a schematic part cross-sectional view illustrating someembodiments of a bioreactor system disclosed herein comprising abioreactor having a cylindrical shape including a perforated barrier;

FIGS. 4A-4I are schematic cross-sectional views illustrating someembodiments of shapes of bioreactors comprising a perforated barrier(12); FIG. 4A presents a bioreactor (300) that has a shape that has acylindrical part (304A) and a frustoconical part (304B); FIG. 4Bpresents a bioreactor (310) that has a shape that has a cylindrical part(314A) and a tapering part (314B); FIG. 4C presents another embodimentof a bioreactor (320) that has a shape that has a cylindrical part(324A) and a tapering part (324B); FIG. 4D presents a bioreactor (330)that has a tapering shape; FIG. 4E presents another embodiment of abioreactor (340) that has a tapering shape; FIG. 4F presents abioreactor (350) that has a shape that has a conical part (354A) and afrustoconical part (354B); FIG. 4G presents a bioreactor (360) that hasa cylindrical shape; FIG. 4H presents a bioreactor (370) that has ashape similar to a chalice, comprising a first chamber (374A) shaped asa hemispherical and a second chamber (374B) shaped as a frustoconicalpart; FIG. 4I presents a bioreactor (380) that comprises a vertical wallportion (380H) and a slanted wall portion (380E);

FIG. 4J is a schematic top view of the bioreactor (380) illustrated inFIG. 4I;

FIG. 5 is a schematic block diagram illustrating the components of abioreactor system (400), in accordance with some embodiments of thebioreactor systems disclosed herein;

FIGS. 6A and 6B are schematic part cross-sectional views illustratingtwo embodiments of possible positional states of a tiltable bioreactor(510); In FIG. 6A, the bioreactor (510) is in a vertical state; In FIG.6B, the bioreactor (510) is in a tilted state;

FIGS. 6C and 6D are schematic part cross-sectional views illustratingtwo embodiments of a bioreactor (550) having a fixed slanted perforatedbarrier;

FIGS. 7-9 are schematic part cross-sectional views illustrating threedifferent embodiments of bioreactors (610, 710, and 810, respectively)including three different types of non-planar (not flat) perforatedbarriers (612, 712, and 812, respectively);

FIGS. 10A and 10B are schematic part cross-sectional views illustratingtwo embodiments of different states of a bioreactor (910) including adeformable perforated barrier (912);

FIGS. 11A and 11B are schematic part cross-sectional views illustratingtwo embodiments of different states of a bioreactor (1010) including abuckling perforated barrier (1012);

FIGS. 12A and 12B are schematic part cross-sectional views illustratingtwo embodiments of different operational states of a bioreactor (1110)including a tiltable perforated barrier (1112), in accordance with someembodiments of the bioreactors of the present application;

FIG. 13 is a schematic part cross-sectional view illustrating anembodiment of a bioreactor system (1250) comprising a bioreactor (10)having a perforated barrier (12) and a cell carrier matrix (1260);

FIGS. 14A-14C show a schematic of an embodiment of a bioreactor used forculturing cells (FIG. 14A) and the growth curves (number of cells versusdays) of cells grown using the bioreactor of FIG. 14A; FIG. 14B showsgrowth curves after 5 days in the T75 flask (Blue line) and theBioreactor (orange); FIG. 14C shows growth curves after 14 days ofgrowth in the bioreactor (yellow line) in comparison with cells grown inT75 flasks, with (blue line) and without (grey line) change of media;

FIGS. 15A-15D present embodiments of processing of cells grown in abioreactor; FIG. 15A presents an embodiment of replacing one liquid withanother, for example replacing growth media with wash buffer; FIG. 15Bpresents another embodiment of replacing one liquid with another,wherein the bioreactor comprises a second barrier (barrier 2) located ina position within a second (upper chamber) above the level of the cells;The bioreactor vessel shown in FIG. 15B is inverted in the image; FIGS.15C and 15D show representative diagrams of a bioreactor constructed oftwo frusto-conical parts, divided into three chambers by two perforatedbarriers, where FIG. 15C demonstrates the bioreactor during cell growthstage and FIG. 15D demonstrates the bioreactor ad its flipped positionduring a washing stage; and.

FIG. 16 is a schematic cross-sectional illustrating a perforated barrierconfigured to control fluid velocity.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements can be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the bioreactorsdescribed herein, and use thereof. In other instances, well-knownmethods, procedures, and components have not been described in detail soas not to obscure the bioreactors described herein and uses thereof.

The present application discloses a cell culturing processing andmanipulating system including bioreactors and bioreactor systemsdesigned for culturing of cells and microorganisms in changing densitiesand adaptive culture volumes starting from isolation to finalformulation. The bioreactors disclosed herein are configured tocontinuously allow all the necessary steps of selecting, culturing,modifying, activating, expanding, washing, concentrating and formulatingin one single unit. According to some embodiments, the bioreactors canbe used in a batch mode, fed batch mode and perfusion mode and can befully controlled in a closed, aseptic environment and can be implementedfor a single use (to be disposed after one culturing cycle) as well asfor multiple cycle uses.

Before explaining the various embodiments of the bioreactors and systemsthereof as disclosed herein in detail, it is noted that the bioreactorsand systems thereof disclosed, are not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples. The bioreactors andsystems thereof disclosed herein can encompass other embodiments or ofbeing practiced or carried out in various ways.

The present application in some embodiments thereof, discloses a flow ora stream of a “medium”, “liquid”, “gas”, “wash buffer”, “solution” or“fluid”. A skilled artisan would appreciate that these terms arealternatively used and having a characteristic of a substance thatcontinually deforms (flows) under an applied pressure and/or an appliedshear stress.

The present application in some embodiments thereof, disclosesbioreactors for growing living cells or microorganisms, and methodsthereof for growing cells or microorganisms in these bioreactorsincluding all culturing steps from isolation to final formulation.

A skilled artisan would appreciate that the terms “cell” and “cells” mayencompass any living cells. In some embodiments, cells that may be grownin a bioreactor disclosed herein comprise any prokaryotic or eukaryoticcell. In some embodiments, cells that may be grown in a bioreactordisclosed herein comprise unicellular and multicellular microorganisms,for example bacteria, archaebacteria, viruses, yeast cells, plant cells,or insect cells.

In some embodiments, eukaryotic cells comprise plant cells, insectcells, animal cells, or fungi. In some embodiments, cells comprisetissue culture cells, primary cells, or reproductive cells. In someembodiments, tissue culture cells or primary cells comprise stem cells,adult cells, transdifferentiated cells, dedifferentiated cells, ordifferentiated cells. In some embodiments, animal cells comprisemammalian cells. For example, mammalian cells may comprise cellsoriginating from a baboon, buffalo, cat, chicken, cow, dog, goat, guineapig, hamster, horse, human, monkey, mouse, pig, quail, or rabbit. Insome embodiments, mammalian cells comprise primary cells comprising stemcells, embryonic cells, adult cells, transdifferentiated cells,dedifferentiated cells, or differentiated cells. In some embodiments,mammalian cells comprise tissue culture cells comprising stem cells,embryonic cells, adult cells, transdifferentiated cells,dedifferentiated cells, or differentiated cells.

In some embodiments, the cell types compatible with growth in abioreactor disclosed herein include stem cells, Acinar cells,Adipocytes, Alveolar cells, Ameloblasts, Annulus Fibrosus Cells,Arachnoidal cells, Astrocytes, Blastoderms, Calvarial Cells, Cancerouscells (Adenocarcinomas, Fibrosarcomas, Glioblastomas, Hepatomas,Melanomas, Myeloid Leukemias, Neuroblastomas, Osteosarcomas, Sarcomas)Cardiomyocytes, Chondrocytes, Chordoma Cells, Chromaffin Cells, CumulusCells, Endothelial cells, Endothelial-like cells, Ensheathing cells,Epithelial cells, Fibroblasts, Fibroblast-like cells, Germ cells,Hepatocytes, Hybridomas, Insulin producing cells, Intersticial Cells,Islets, Keratinocytes, Lymphocytic cells, Macrophages, Mast cells,Melanocytes, Meniscus Cells, Mesangial cells, Mesenchymal PrecursorCells, Monocytes, Mononuclear Cells, Myeloblasts, Myoblasts,Myofibroblasts, Neuronal cells, Nucleus cells, Odontoblasts, Oocytes,Osteoblasts, Osteoblast-like cells, Osteoclasts, Osteoclast precursorcells, Oval Cells, Papilla cells, Parenchymal cells, Pericytess,Peridontal Ligament Cells, Periosteal cells, Platelets, Pneumocytes,Preadipocytes, Proepicardium cells, Renal cells, Salisphere cells,Schwann cells, Secretory cells, Smooth Muscle cells, Sperm cells,Stellate Cells, Stem Cells, Stem Cell-like cells, Stertoli Cells,Stromal cells, Synovial cells, Synoviocytes, T Cells, Tenocytes,T-lymphoblasts, Trophoblasts, Natural killer cells, dendritic cells,Urothelial cells, Vitreous cells, and the like; the cells originatingfrom, for example and without limitation, any of the following tissues:Adipose Tissue, Adrenal gland, Amniotic fluid, Amniotic sac, Aorta,Artery (Carotid, Coronary, Pulmonary), Bile Duct, Bladder, Blood, Bone,Bone Marrow, Brain (including Cerebral Cortex), Breast, Bronchi,Cartilage, Cervix, Chorionic Villi, Colon, Conjunctiva, ConnectiveTissue, Cornea, Dental Pulp, Duodenum, Dura Mater, Ear, Endometrioticcyst, Endometrium, Esophagus, Eye, Foreskin, Gallbladder, Ganglia,Gingiva, Head/Neck, Heart, Heart Valve, Hippocampus, Iliac,Intervertebral Disc, Joint, Jugular vein, Kidney, Knee, Lacrimal Gland,Ligament, Liver, Lung, Lymph node, Mammary gland, Mandible, Meninges,Mesoderm, Microvasculature, Mucosa, Muscle-derived (MD), MyeloidLeukemia, Myeloma, Nasal, Nasopharyngeal, Nerve, Nucleus Pulposus, OralMucosa, Ovary, Pancreas, Parotid Gland, Penis, Placenta, Prostate,Renal, Respiratory Tract, Retina, Salivary Gland, Saphenous Vein,Sciatic Nerve, Skeletal Muscle, Skin, Small Intestine, Sphincter, Spine,Spleen, Stomach, Synovium, Teeth, Tendon, Testes, Thyroid, Tonsil,Trachea, Umbilical Artery, Umbilical Cord, Umbilical Cord Blood,Umbilical Cord Vein, Umbilical Cord (Wartons Jelly), Urinary tract,Uterus, Vasculature, Ventricle, Vocal folds and cells, or anycombination thereof. In some embodiments, the cells grown in abioreactor disclosed herein may comprise a combination of different celltypes. As used herein, in some embodiments the terms “cells” and“microorganisms” may be used interchangeably having all the samemeanings and qualities.

In some embodiments, the products of the cells or microorganisms grownin a bioreactor disclosed herein are collected, for example proteins,peptides, antibiotics or amino acids. In some embodiments, any productof a cell or microorganism grown in a large-scale manner in a bioreactordisclosed herein and synthesized by the cell or microorganism, can becollected.

The bioreactors disclosed in the present application, non-limiting ofwhich are presented in FIG. 1 (10), FIG. 2 (110), FIG. 3 (210), FIG. 4A(300), FIG. 4B (310), FIG. 4C (320), FIG. 4D (330), FIG. 4E (340), FIG.4F (350), FIG. 4G (360), FIG. 4H (370), FIG. 4I (380), FIGS. 6A and 6B(510), FIGS. 6C and 6D (550), FIG. 7 (610), FIG. 8 (710), FIG. 9 (810),FIGS. 10A and 10B (910), FIGS. 11A and 11B (1010), FIGS. 12A and 12B(1110), and FIG. 14A, can be shaped like a hollow vessel including aperforated barrier that divides the internal volume or space within thevessel into a first (lower) chamber and a second (upper) chamberdisposed above the first chamber.

According to some embodiments, a bioreactor described herein for growingcells or microorganisms therein, the bioreactor comprising:

-   -   a closed vessel enclosing a space therein;    -   a barrier having a plurality of perforations therein, the        barrier is sealingly disposed within the space configured to        divide the space into a first chamber and a second chamber,        wherein the second chamber is configured to accommodate the        growing cells or microorganisms therein, and wherein a diameter        of the perforations is configured to allow a fluid flow solely        between the first chamber and the second chamber and vice versa,    -   one or more fluid inlet ports for introducing the fluid into the        first chamber; and    -   one or more fluid outlet ports for allowing the fluid to exit        from the second chamber.

According to some embodiments, the bioreactor vessel can be constructedof at least two parts. And according to some embodiments, the barriercan be attached between the two parts. According to some embodiments,more perforated barriers can be provided, in some cases between thedifferent parts of the vessel. According to some embodiments, thebarrier is disposed in contact with walls of the vessel (as demonstratedin FIGS. 1-4, 6-13, 15-16).

According to some embodiments, the first chamber is a lower chamber andthe second chamber is an upper chamber and wherein the fluid flow is anupstream flow from the lower chamber towards the upper chamber (againstgravity direction).

Without being limiting, in some embodiments, a bioreactor comprises achamber comprising a widening shape, for example a conical frustumshape, or a portion thereof, which is configured to lead to reduction ofvelocity of a fluid. In some embodiments, a bioreactor comprises achamber of two parts divided by a perforated barrier, wherein the batherallows a constant fluid flow, for example but not limited to a fluidgrowth media, and wherein the cells are retained in the second (upper)chamber. In some embodiments, a bioreactor comprises reduced velocity offlow of a fluid in the second (upper) chamber and a uniform and gentleflow of a fluid throughout the vessel. In some embodiments, the gentleand uniform flow combined with the reduced velocity in the second(upper) chamber results in a balance between the mass of cells (cellmass) and the velocity of the fluid resulting in a steady mass of cellsknown as a “floating cake”. In some embodiments, a floating cake ofcells localized to the lower portion of the second (upper) chamber.

In some embodiments, use of a bioreactor described herein results in aconstant fluid flow. In some embodiments, use of a bioreactor results ina constant flow of growth media and cell feeding during the culturingprocess. In some embodiments, a fluid, for example a growth media, canbe exchanged during culturing, wherein very small volumes and/or verylarge volumes provide the for adaptive and optimal cell feeding. In someembodiments, use of a bioreactor described herein comprises cell washingand harvesting to a selected media in a very gentle and efficient mannerwithout the need to open the bioreactor chamber. In some embodiments,use of a bioreactor described herein provides for optimal and adaptiveculturing, wherein manipulation of cells or microorganisms is performedin a closed system, wherein the manipulation can be automated, andwherein cells experience minimal sheer force. In some embodiments, useof a bioreactor described herein supports high density growth of cellsor microorganisms. In some embodiments, the density achieved, by thebioreactors disclosed herein, can be greater than 10-fold that observedusing standard culturing conditions.

A skilled artisan would appreciate that the term “perforated barrier”may be used interchangeable with the term “filter” or “membrane” or“perforated plate” having all the same qualities and meanings.

In some embodiments, the perforated barrier comprises a plurality ofperforations therein that is configured to allow bidirectional flow of aliquid, for example a growth media through the perforations of theperforated barrier such that liquid can flow from the first chamber tothe second chamber and also from the second chamber to the firstchamber.

A skilled artisan would appreciate that the term “first chamber” as usedherein, may in some embodiments be used interchangeable with the term“lower chamber” having all the same meanings and qualities thereof. Askilled artisan would appreciate that the term “second chamber” as usedherein, may in some embodiments be used interchangeable with the term“upper chamber” having all the same meanings and qualities thereof. Insome embodiments, cells are cultured in the second chamber of bioreactorvessel.

In some embodiments, the perforated barrier is configured to allowbidirectional flow of liquid including additional factors through theperforations of the perforated barrier such that liquid and additionalfactor or factors can flow from the first chamber to the second chamberand from the second chamber to the first chamber. In some embodiments,the perforation diameter is configured to allow liquid flow solely fromthe first chamber to the second chamber and from the second chamber tothe first chamber. In some embodiments, the perforation diameter isconfigured to allow liquid including a factor or factors to flow solelyfrom the first chamber to the second chamber and from the second chamberto the first chamber. In some embodiments, the factor or factors doesnot include cells or microorganisms. In some embodiments, the perforatedbarrier comprising a plurality of perforations, which do not allow cellsor microorganisms grown in the vessel of the bioreactor to pass throughthe perforated barrier.

A skilled artisan would appreciate that flow may encompass flow of aliquid fluid comprising a growth media, a washing solution, a nutrientsolution, a selection solution, an enzyme mixture solution, a collectionsolution, a final formulation solution, a storage solution, or anycombination thereof. In some embodiments, a liquid comprises a growthmedia, a washing solution, a nutrient solution, a collection solution, aharvesting solution, a storage solution, or any combination thereof. Insome embodiments, a liquid comprises additional factors, whereinnon-limiting examples of factors that may be added include nutrients,gasses, activation factors, induction factors, antibiotics, antifungalagents, and salts. In some embodiments, any factor beneficial for thegrowth and collection of cells or microorganisms in bioreactor systemsdescribed herein may be added to a liquid. In some embodiments, a factordissolves within the liquid, wherein the liquid represents a solvent andthe factor a solute to form a solution. In some embodiments, a factorremains as a particulate within the liquid.

A skilled artisan would appreciate that the term “plurality” mayencompass the number of perforations (pores) in a perforated barrier. Insome embodiments, the plurality of perforations is determined based on aneeded rate of exchange of media or other liquid flowing from a firstchamber to a second chamber, or from a second chamber to a firstchamber. In some embodiments, the plurality of perforations isdetermined based on the flow rate of media or other liquid flowing froma first chamber to a second chamber, or from a second chamber to a firstchamber. In some embodiments, the plurality of perforations isdetermined based on the pattern of flow of media or other liquid flowingfrom a first chamber to a second chamber, or from a second chamber to afirst chamber.

In some embodiments, the arrangement of perforations within a perforatedbarrier is configured to affect the pattern of flow of a media or otherliquid flowing from a first chamber to a second chamber, or from asecond chamber to a first chamber. In some embodiments, a perforatedbarrier comprises an evenly spaced plurality of perforations. In someembodiments, a perforated barrier comprises an uneven spacing of aplurality of perforations.

In some embodiments, the mean perforation diameter or effective meandiameter of the perforations in the perforated barrier is selected suchthat it does not allow cells or microorganisms grown in the bioreactorto pass through the perforated barrier. For example, in someembodiments, determining of the size of a perforation diameter comprisesmeasuring a cell or microorganism size and determining a cell ormicroorganism shape, choosing a perforation diameter (perforation poresize) that would prevent the cell or microorganism from passing througha perforated barrier having the chosen pore size.

According to some related embodiments, the mean perforation diameter oreffective mean diameter of the perforations in the perforated barrier isselected to be smaller than: 120 micrometer, or 100 micrometer or, 75micrometer, or 50 micrometer, or 25 micrometer, or 15 micrometer.According to some related embodiments, the mean perforation diameter oreffective mean diameter of the perforations in the perforated barrier isselected to be larger than: 0.1 micrometer, or 0.2 micrometer, or 0.3micro meter or, 0.45 micrometer, or 0.75 micrometer or, 1.0 micrometer.According to some related embodiments, the mean perforation diameter oreffective mean diameter of the perforations in the perforated barrier isselected between 0.1 micrometer and 120 micrometer. According to somerelated embodiments, the mean perforation diameter or the effective meandiameter of the perforations in the perforated barrier does not allowcells or microorganisms to pass from one chamber to a second chamber.For example, the mean perforation diameter or the effective meandiameter of the perforations in the perforated barrier is selected sothat cells or microorganisms grown in an upper chamber may not pass intothe lower chamber.

In some embodiments, the cell or microorganism have a spherical shape,accordingly the diameter of the cell or microorganism is used indetermining perforation size. In some embodiments, the cell ormicroorganism may not have a spherical shape. In some embodiments, acell or a microorganism may comprise a non-symmetrical shape, forexample but in no way limiting a rod shape. Wherein a cell or amicroorganism has a non-symmetrical shape, measurement for determiningpore size would be based on the smallest diameter presented by a cell.In some embodiments, a cell may have the capacity to change shapes.Wherein a cell or a microorganism has the capacity to change shape,measurement for determining pore size would be based on the smallestdiameter presented by the cell or microorganism that would allow passageof a cell or microorganism through a pore. In some embodiments, a cellor a microorganism may be deformable. Wherein a cell or a microorganismis deformable, cell size determination takes into account the diameterof the deformed cell or microorganism.

In some embodiments, a plurality of perforations comprises perforationsof all the same size. In some embodiments, a plurality of perforationcomprises perforations that are not all the same size. In someembodiments, perforations of different sizes comprise a randomdistribution. In some embodiments, the distribution of perforations ofdifferent sizes is determined based on fluid flow patterns from the flowof a liquid from a first chamber to the second chamber and from thesecond chamber to the first chamber.

In some embodiments, the shape of the perforations is symmetrical. Insome embodiments, the shape of the perforations is non-symmetrical. Insome embodiments, the shape of the perforation comprises a circularshape, an irregular in shape, an elliptical shape, or a polygonal. Insome embodiment, a plurality of perforations comprises perforations allof the same shape. In some embodiment, a plurality of perforationscomprises perforations of different shapes.

In some embodiments, the mean perforation diameter or effective meandiameter of the perforations in the perforated barrier is determined byselecting a diameter configured to allow the flow of a liquid from afirst chamber to the second chamber and also from the second chamber tothe first chamber, and does not allow cells or microorganisms grown inthe bioreactor to pass through the perforated barrier. In someembodiments, the mean perforation diameter or effective mean diameter ofthe perforations in the perforated barrier is determined by selecting adiameter that allows for the flow of a liquid comprising additionalfactors from a first chamber to the second chamber and also from thesecond chamber to the first chamber, and does not allow cells ormicroorganisms grown in the bioreactor to pass through the perforatedbarrier. In some embodiments, the mean perforation diameter or effectivemean diameter of the perforations in the perforated barrier isdetermined by selecting a diameter that allows for the flow of a liquidcomprising additional factors and products produced from the cells ormicroorganisms from a first chamber to the second chamber and also fromthe second chamber to the first chamber, and does not allow cells ormicroorganisms grown in the bioreactor to pass through the perforatedbarrier.

In some embodiments, the perforation diameter (pore size) or effectivemean diameter comprises about 0.1 to 40 micrometer. In some embodiments,the perforation diameter (pore size) or effective mean diametercomprises about 0.2 to 10 micrometer. In some embodiments, theperforation diameter (pore size) or effective mean diameter comprisesabout 10 to 40 micrometer. In some embodiments, the perforation diameter(pore size) or effective mean diameter is larger than 40 micrometers. Insome embodiments, the perforation diameter (pore size) or effective meandiameter comprises about 40 to 60 micrometer. In some embodiments, theperforation diameter (pore size) or effective mean diameter comprisesabout 60 to 100 micrometer.

In some embodiments, the perforation diameter (pore size) or effectivemean diameter is configured to prevent cells or microorganisms, to flowthrough the pore. In some embodiments, the perforation diameter oreffective mean diameter is configured to prevent cells or microorganismsbound to beads to flow through the pore. In some embodiments, the porediameter, of the perforations of a perforated barrier having a pluralityof perforations therein, is configured to allow solely liquid flow fromthe first chamber to the second chamber and from the second chamber tothe first chamber. In some embodiments the liquid can comprise solutesand/or added factors. In some embodiments, the pore diameter of theperforations of a perforated barrier having a plurality of perforationstherein, is configured to allow solely liquid flow from the firstchamber to the second chamber and from the second chamber to the firstchamber, wherein the pore diameter is configure to not allow the passageof cells or microorganisms from the first chamber to the second chamberand from the second chamber to the first chamber.

In some embodiments, the perforated barrier is configured and useful,for example, in confining the grown cells to the second chamber withinthe reactor and in harvesting the cells. According to some embodiments,the present application also discloses bioreactor systems including thebioreactors and methods for growing cells or microorganisms in thebioreactors and bioreactor systems from isolation to final formulation.

In some embodiments, a bioreactor comprises an additional lowerperforated barrier 12D below the perforated barrier 12 (which is presentat the bottom of the upper chamber); see for example FIG. 1 (12) whereinthe perforated barrier 12 comprises the bottom of the upper chamber. Insome embodiments, the additional lower perforated barrier 12D is locatedbetween the bottom surface of the vessel (at the lower chamber) and theperforated barrier 12 (which is forming the bottom surface of the upperchamber); for example between 10B and 12 of FIG. 1. In some embodiments,the upstream flow of liquid from a lower chamber to an upper chamberpasses through the two perforated barriers 12 and 12D. The additionallower perforated barrier 12D is configured to assist in aligning theflow of a liquid (straitening, providing linearity and uniform flowthereto) before it reaches the perforated barrier 12 that comprises thebottom of the upper chamber. This arrangement is configured to improvethe linearity (and uniformity) of a liquid's flow. According to someembodiments, aligning the stream comprises providing an approximatelyeven longitudinal flow rate along different radial locations of theperforated barrier [v(r₁)≈v(r₂)], or in other words the flow rate issubstantially equal at every distance of the geometrical center of theperforated barrier. According to some embodiments the lower perforatedbarrier is sealingly attached to the walls of the lower chamber, andwherein its pores size is configured the prevent passage of cells ormicroorganism. According to some embodiments, both the perforatedbarrier 12 and the lower perforated barrier 12D are configured to alignthe liquid flow rate. According to some related embodiments, the meanperforation diameter or effective mean diameter of the perforations inthe lower perforated barrier 12D is selected between 0.1 micrometer and1 millimeter.

According to some embodiments, the lower perforated barrier 12D isconfigured to control the fluid velocity. A non limiting example forsuch a velocity controlling barrier 1600 is detailed in FIG. 16. Asdemonstrated in FIG. 16, the pores 1601 of a velocity controllingperforated barrier 1600 can comprise conical shapes; conical shape ofthe pores can be similar or different between the different pores, somepores can be similar and some can be different. According to someembodiments, the wider base of the conical pores is located at thebottom side of the barrier; such a configuration can provide the flowwith an increasing flow rate towards the upper side of the barrier.According to some embodiments, pore/s 1602 closer to the center of thebarrier can have a wider cone, or a wider opening at the upper side ofthe barrier, than of the peripheral pores 1601; such a configuration canprovide an approximately even longitudinal flow rate along the differentradial locations [v(r₁)≈v(r₂)] of the perforated barrier 1600. Accordingto such embodiments, a fluid impeller may not be required.

In some embodiments, the presences of the additional lower perforatedbarrier 12D is configured to trap air bubbles, air clusters, and debriswhich would otherwise clog and block flow through perforations of theupper perforated barrier 12 and interfere with the linearity anduniformity of flow.

In some embodiments, a bioreactor comprises an additional screeningperforated barrier 1502 above the perforated barrier (first perforatedbarrier) 1512 (which is present at the bottom of the upper chamber), thescreening perforated barrier is disposed sealingly to the walls of theupper chamber. FIG. 15A demonstrates the first perforated barrier 1512and the additional screening perforated barrier 1502, which ispositioned above the level of the cells mass 3. According to someembodiments, the additional screening perforated barrier 1502 isconfigured to prevent cells or microorganism passage for example toprevent the cells from leaving the bioreactor. In some embodiments, thebioreactor vessel is in an inverted position (See also Example 3 below)the flow of liquid is downstream 1520 (approximately with gravitydirection) from an upper (the second 1540) chamber to a lower (the first1550) chamber. This configuration is configured in some embodiments tobe used during washing of cells or exchange of media or liquid solutionsallowing wider surface area barrier, which enables to reduce a cloggingof the barrier by the cell mass.

According to related embodiments, the bioreactor comprises, threeperforated barriers:

-   -   a primary perforated barrier 1512 (FIG. 15A), configured to        separate between the upper and the lower chambers (1540,1550) of        the bioreactor's vessel and to prevent passage of cells and        microorganism there between;    -   an upper perforated barrier 1502 (FIG. 15A), located in the        upper chamber 1540 above cell mass 3 configured to prevent        passage of cells and microorganism; therefore cell mass is kept        between the primary and the upper perforated barriers        (1512,1502) and;    -   a lower perforated barrier 12D (FIG. 1), located in the first        chamber 14A below primary perforated barrier 12, configured to        align and/or control the fluid flow before reaching to the        primary perforated barrier 12.

According to some related embodiments, the primary and the upperperforated barriers (1512, 1502, FIG. 15A) comprise similar pores sizeconfigured to prevent passage of cells or microorganisms.

According to some embodiments, the size of the pores of the lowerperforated barrier (12D, FIG. 1) can be similar to—or can be differentthan—the size of the pores of the primary and the upper barriers(1512,1502, FIG. 15A).

One skilled in the art would appreciate that the range, shape, anddistribution of pores may be similar or different between the differentperforated barriers. In some embodiments, the diameter or effectivediameter of the perforations (pores) of an additional perforated barriercomprise different sizes of pores than is present in the perforatedbarrier that separates the first and second chambers. In someembodiments, the diameter or effective diameter of the perforations(pores) of an additional perforated barrier comprise similar sizes ofpores than perforated barrier that separates the first and secondchambers. In some embodiments, the shape of the perforations (pores) ofan additional perforated barrier comprises different shapes of poresthan is present in the perforated barrier that separates the first andsecond chambers. In some embodiments, the shape of the perforations(pores) of an additional perforated barrier comprises similar shapes ofpores than the perforated barrier that separates the first and secondchambers. In some embodiments, the distribution of the perforations(pores) of an additional perforated barrier comprises differentdistribution of pores than is present in the perforated barrier thatseparates the first and second chambers. In some embodiments, thedistribution of the perforations (pores) of an additional perforatedbarrier comprises similar distribution of pores than the perforatedbarrier that separates the first and second chambers.

In some embodiments, a bioreactor comprises an additional barrier withthe second chamber above the cells and an additional barrier within thefirst chamber below the barrier that separates the first and secondchambers.

One skilled in the art would appreciate that the surface area of anadditional perforated barrier can be greater than or less than thesurface area of the barrier that separates the first chamber from thesecond chamber. In some embodiments, an additional perforated barrierhas a larger surface area than the surface area of the barrier thatseparates the first chamber from the second chamber. In someembodiments, an additional perforated barrier has a smaller surface areathan the surface area of the barrier that separates the first chamberfrom the second chamber.

The disclosed bioreactors and bioreactor systems allows growing,processing and formulating the cells or other microorganisms in oneclosed single or multiple use system minimizing the risk ofcontamination and allowing efficient processing. According to someembodiments, bioreactors disclosed herein are configured to allowgrowing cells or other microorganisms to a desired concentration. In oneembodiment, bioreactors disclosed herein provide a sterile environment.In one embodiment, bioreactor systems disclosed herein provide a sterileenvironment. Furthermore, as the cells or microorganisms are culturedand propagated they require more media and nutrients and largerculturing volumes. Some embodiments of the bioreactors describedhereinafter include adaptive controlled volume changes (variablebioreactor volume) and media refreshment without the need to transferthe cells or microorganisms to a larger container.

In some embodiments, the bioreactors of the present application areconfigured to be used for growing non-adherent cells, which aresuspended in the growth medium. In some embodiments, the bioreactorsdisclosed herein are configured to be used for growing adherent cells byincluding or adding a suitable cell supporting matrix into the secondchamber of the bioreactor. The cell supporting matrix can be any type ofcell supporting matrix known in the art to which the cells can adhere.If such a cell supporting matrix is being used in the bioreactor, it maybe necessary to detach the cells from the cell supporting matrix byusing detachment methods known in the art. As used herein, in someembodiments, the terms “cell supporting matrix” and “cell carriermatrix” and conjugates thereof may be used interchangeably having allthe same meanings and qualities.

The bioreactors of the present application are configured to have afixed volume or a variable volume. A skilled artisan would appreciatethat in some embodiments, the terms “bioreactor” and “vessel” may beused interchangeable having all the same meanings and qualities. Inembodiments wherein the bioreactor comprises a fixed volume, the rate offlow of a liquid, for example a growth medium can be controlled but thelevel and volume of the liquid, for example a growth medium in thebioreactor is substantially fixed. In embodiments wherein the bioreactorcomprises a variable volume, the rate of flow of the liquid, for examplea growth medium can be controlled and the level and volume of growthmedium in the bioreactor can be variable. In some embodiments, variablethe liquid levels, for example growth medium levels can be achieved byusing multiple fluid outlet ports opening into the second chamber of thebioreactor at various different heights along the length of the walls ofthe bioreactor. A non-limiting example of this is presented in FIG. 2.

In some embodiments, the working volume of media is low, wherein cellsare grown to high density cultures. In some embodiments, wherein theworking volume is low, the rate of flow is also low or no flow at all.In some embodiments, the flow rate is low. In some embodiments, there isno flow from a first chamber to the second or from the second chamber tothe first. In some embodiments, there is no flow from a first chamber tothe second and from the second chamber to the first. In someembodiments, wherein the working volume is low, the medium is optimizedfor high density growth of cells. In some embodiments, wherein theworking volume is low, cell growth is optimized for higher yields andlower media needs than are achievable in other bioreactors.

In some embodiments, when a culture comprises a small number of cells,for example less than the maximal number of cells that can be culturedin a bioreactor described herein, the cells are cultured in a low volumeof growth media, as cells proliferate and the number of cells increasesthe volume within the chamber comprising the cells can be increased. Ata point a flow cycle can be implemented, wherein the flow of liquid, forexample growth media, increases as the quantity of cells increases. Insome embodiments, nutrients can be added to the liquid, e.g., a growthmedia based on cell growth needs. In some embodiments, culturing cellsin a bioreactor described herein maintains cells within a cell densityrange by adjusting the volume of liquid, e.g., growth media, within thebioreactor. In some embodiments, use of a flow cycle as described hereinresults in lower growth media needs for culturing an equivalent numberof cells. In some embodiments, a flow cycle is used in a bioreactordescribed herein, wherein the supply of a growth media is regulatedbased on cells' needs. In other words, cells are fed only as needed. Insome embodiments, the flow cycle controls the proliferation rate ofcells.

According to some embodiments, each of the multiple outlet ports areconfigured to have a valve therein and configured be connected anddisconnected fluidically to a common manifold feeding a pump. The levelof a liquid, e.g., a growth medium in the bioreactor of such embodimentscan be varied by suitably opening the valve of a selected fluid outletport and closing all the valves of the remaining fluid outlet ports.According to some embodiments, controlling the volume of a liquid, e.g.,a growth medium in the bioreactor advantageously allows expanding theculture as the cells continue to proliferate without opening thebioreactor and without the need of using methods used in otherbioreactor systems, such as, for example cell passaging anddish/container replacement.

In some embodiments, the bioreactors are configured to include a fluidimpeller or fluid disperser disposed in the first (lower) chamber of thebioreactor's vessel. In some embodiments, the bioreactor is configuredto include an oxygenating system for oxygenating the growth medium.

Bubbles may in certain embodiments be created by the oxygenating system.Bubbles in a lower chamber may in some embodiments, have a negativeimpact on a bioreactor, as the bubbles may stick to a perforated barrierand interfere with the flow of liquid from one chamber to the nextchamber. Additionally, nano bubbles that pass through the perforationsof the barrier tend to lift cells up, which may interfere with the highdensity growth of a floating cell cake.

According to some embodiments, the lower perforated 12D (FIG. 1) isconfigured to prevent passage of bubbles created or formed in the lowerchamber from reaching and blocking the perforated barrier 12; bubblescreated for example by the oxygenating system. According to someembodiments, bubbles with an approximate diameter of several nanometersdo pass the lower perforated barrier 12D and the perforated barrier 12and assist in lifting the cells or microorganism up the liquid's flow.

According to some embodiments, the bioreactors disclosed herein areconfigured to have various different shapes and at least the portions ofthe walls of the bioreactors, which define the second chamber isconfigured to be straight (vertical) or configured to be slanted at anangle to the vertical (or slanted with respect to a longitudinal axis ofthe bioreactor). In some embodiments, some of the walls surrounding thesecond chambers are configured to be vertical and some of the walls areconfigured to be slanted. Non-limiting examples of shapes of thebioreactor vessel are presented in FIG. 4A (304A and 304B), FIG. 4B(314A and 314B), FIG. 4C (324A and 324B), FIG. 4D (334A and 334B), FIG.4E (344A and 344B), FIG. 4F (354A and 354B), FIG. 4G (364A and 364B),FIG. 4H (374A and 374B), FIG. 4I (384A and 308B).

The upward increasing transversal cross-sectional area of the secondchamber in such embodiments is configured to allow a fluid velocitygradient to be established along the vertical direction (along thelongitudinal axis of the bioreactor), such that the growth medium flowvelocity decreases with increasing transversal cross-sectional area.According to some embodiments, this flow velocity gradient combined withthe gravitational force acting on the cells suspended in the growthmedium assists in suspending the cells at some desired region within thevolume of growth medium contained in the second chamber. In someembodiments, regulation of flow rates of medium maintains cells in adesired position within a bioreactor. In some embodiments, regulation offlow rates of medium maintains cells in a desired position within abioreactor. In some embodiments, regulation of flow rates in relation tothe radius of the bioreactor, or chamber thereof, of medium maintainscells in a desired position within a bioreactor.

In some embodiments, the desired position is lower than the exit port.For example see FIG. 1, if cells suspended within a liquid rise withinthe upper chamber at a flow rate of 1 mm per min (middle set of arrows37B), in the lower part there can be for example a flow rate of 3 mm permin (arrows at the level of the barrier 37A), in the middle a flow rateof 1 mm per min (37B), and a few cm above were the media exits thechamber via a port/valve the flow rate can be 0.2 mm per min (would beabove upper set of arrows 37C and above the level of the exit port 26).In some embodiments, the position of the cells is determined by the flowrate. In some embodiments, the position of the cells is lower than theexit port. A position for cells lower than the exit port can be desiredwhen washing cells, when removing sub-populations of cells, whenexchanging a liquid, when adding factors, or any combination thereof.

A skilled artisan would appreciate that a cell population may comprisecells of different sizes, charge, and mass. In some embodiments, cellscan be separated within different positions within a bioreactordisclosed herein, based on cell characteristics including size, charge,and mass. In some embodiments, cells are maintained within differentpositions within a bioreactor disclosed herein based on cellcharacteristics including size, charge, and mass.

A skilled artisan would appreciate that cell size varies based on thetype of cell. For example a red blood cell is about 6-8 mm in diameter,a T-lymphocyte is about 9-12 mm in diameter, a mesenchymal stem cell(MSC) is about 15-21 mm in diameter, and a macrophage is about 50 mm indiameter. The volume between cells can be dramatically different aswell. In some embodiments, a bioreactor system disclosed herein isconfigured to be used to separate blood cells by regulating the flowrate.

In some embodiments, the flow rate comprises a range of about 0.01 mmper minute to 50 mm per minute. In some embodiment, the flow ratecomprises a range of about 0.01 mm/min to 0.1 mm/min. In someembodiment, the flow rate comprises a range of about 0.1 mm/min to 1.0mm/min. In some embodiment, the flow rate comprises a range of about 1.0mm/min to 2.0 mm/min. In some embodiment, the flow rate comprises arange of about 2.0 mm/min to 3.0 mm/min. In some embodiment, the flowrate comprises a range of about 3.0 mm/min to 4.0 mm/min. In someembodiment, the flow rate comprises a range of about 4.0 mm/min to 5.0mm/min. In some embodiment, the flow rate comprises a range of about 5.0mm/min to 10.0 mm/min. In some embodiment, the flow rate comprises arange of about 10 mm/min to 15 mm/min. In some embodiment, the flow ratecomprises a range of about 15 mm/min to 20 mm/min. In some embodiment,the flow rate comprises a range of about 20 mm/min to 25 mm/min. In someembodiment, the flow rate comprises a range of about 25 mm/min to 30mm/min. In some embodiment, the flow rate comprises a range of about 30mm/min to 35 mm/min. In some embodiment, the flow rate comprises a rangeof about 35 mm/min to 40 mm/min. In some embodiment, the flow ratecomprises a range of about 40 mm/min to 45 mm/min. In some embodiment,the flow rate comprises a range of about 45 mm/min to 50 mm/min.

In some embodiments, the flow rate within a bioreactor is different indifferent positions within the bioreactor (See for example FIG. 1 andthe accompanying explanation thereof below, and the representative flowrate arrows 37A, 37B, and 37C, or FIG. 13 and representative flow ratearrows 37A and 37C).

In some embodiments, the size, charge, and/or mass of a population ofcells can be artificially changed. For example, in some embodiments,cells can be cultured with beads, wherein the cells bind to the beadsresulting in cell-bead complexes having a higher mass and differentshape then the cells not attached to beads. In some embodiments, 100% ofcells are bound to a bead. In some embodiments, a sub-set of cells arebound to a bead. In some embodiments, at least 90% of cells, 80% ofcells, 70% of cells, 60% of cells, 50% of cells, 40% of cells, 30% ofcells, 20% of cells, or 10% of cells are bound to a bead. In someembodiments, less than 10% of cells are bound to a bead.

In some embodiments, cells bound to beads are excluded from collectionof the final cell population. In some embodiments, cells bound to beadsare the cells desired to be collected as the final cell population. Forexample, in one embodiment, following addition of beads, wherein asubpopulation of cells binds to the beads in a specific fashion,increasing the flow rate will result in the cells not bound to beadsrising at an increased rate compared with the cells bound to the beads,so these non-bound cells can exit the vessel chamber from an exit portwherein the bound cells remain in a position lower than the exit port.In some embodiments, the non-bound cells are collected upon exiting thebioreactor chamber. In some embodiments, the non-bound cells aredisposed of upon exiting the bioreactor chamber and the bound cells areharvested.

In some embodiments, the surface of beads can comprise an antibody, areceptor ligand, a carbohydrate binding molecule, a lectin, or acomponent of a binding pair for example biotin. In some embodiments, thesurface of beads comprises a positive surface charge. In someembodiments, binding between beads and cells or a subpopulation thereofis reversible. In some embodiments, binding between beads and cells or asubpopulation thereof is irreversible.

In some embodiments, bioreactors are configured to include one or moreharvesting ports that are configured to open into the second chamber atthe vicinity of the perforated barrier, or, alternatively, areconfigured to open at the upper surface of the perforated barrier.Non-limiting examples of harvesting ports that are configured to openinto the second chamber or at the upper surface of the perforatedbarrier are presented in FIG. 1 (21), FIG. 2 (127), FIG. 6A and FIG. 6B(521), FIGS. 6C and 6D (531), FIGS. 7 (627), FIG. 8 (727), FIG. 9 (827),FIGS. 10A and 10B (927), FIGS. 11A and 11B (927), and FIGS. 12A and 12B(1127).

In accordance with some embodiments, the entire reactor or perforatedbarrier are configured to be tiltable at an angle to the vertical toassist the harvesting of the cells. In some embodiments, harvesting ofthe cells, microorganisms, or products thereof, grown in a bioreactordisclosed herein comprises sterile harvesting of the cells,microorganisms, or products thereof. Non-limiting examples of perforatedbarriers are presented in FIG. 1 (12), FIG. 7 (612), FIG. 8 (712), FIG.9 (812), FIGS. 10A and 10B (912), FIGS. 11A and 11B (1012), and FIGS.12A and 12B (1112).

In accordance with some embodiments of the bioreactor, the perforatedbarrier is configured to be a fixed (non-movable) barrier. In someembodiments, a fixed perforated barrier is sealingly attached to thevessel walls. In accordance with some other embodiments, the perforatedbarrier is configured to be a movable and/or tiltable perforatedbarrier. In accordance with some embodiments of the bioreactor fixedperforated barriers is configured to be a flat perforated barrier, aflat perforated barrier inclined at an angle to a longitudinal axis ofthe bioreactor, a concave perforated barrier with a concave uppersurface facing the top of the bioreactor, a tapering perforated barrier,or a conical perforated barrier, or any combination thereof.

In accordance with some embodiments of the bioreactor, the movableperforated barriers are configured to be a movable perforated barriersealingly attached to the vessel walls of the bioreactor by a flexibleand/or stretchable member. The flexible and/or stretchable member issealingly attached to a perimeter of the perforated barrier andsealingly attached to the vessel wall. In accordance with someembodiments of the bioreactor, the movable perforated barrier isconfigured to be a deformable and/or flexible perforated barrier, or aconvex buckling perforated barrier with a convex upper surface facingthe top of the bioreactor.

A skilled artisan would appreciate that the term “sealingly” anddifferent grammatical forms thereof, refers to an attachment between thebarrier and the vessel wall wherein there is no flow through the barrierof any kind of material unless through perforations.

In some embodiments, bioreactor systems including the bioreactors of thepresent application are configured to also include temperature controlsystems, pumps for circulating the growth medium, one or more fluidreservoirs connectable to the bioreactor for introducing volumes ofgrowth medium and/or additives and/or substances required formaintaining the level of nutrients and/or any other materials necessaryfor cell growth.

Other substances required for any steps of growing and/or maintaining,washing, and/or proliferating and/or differentiating and/or activatingand/or detaching the cells for harvesting can also be added through suchfluid reservoirs, including various enzymes, growth factors, activatingfactors, differentiating factors, washing buffers, pH adjustments,dissolved Oxygen adjustments, Nutrients or any other necessarysubstances or compounds. In some embodiments, living cells can also beadded for co-culturing with or activating the cells within thebioreactor. In some embodiments, other substances required for inducingand/or maintaining induction of a cell product or microorganism productcan also be added to medium within the bioreactor.

In some embodiments, bioreactor systems disclosed herein are configuredto also include a controller for controlling the operation of thebioreactor, for opening and/or closing various different valves of thebioreactor, for controlling the flow of growth medium or other fluidsthrough the bioreactor by controlling the pump and/or various differentvalves. As used herein, one skilled in the art would appreciate that theterm “flow velocity” may be used interchangeable with “flow rate” havingall the same meanings and qualities. As used herein, one skilled in theart would appreciate that the term “perforations” may be usedinterchangeable with “pores” having all the same meanings and qualities.

In some embodiments, the flow rate directly or indirectly influences thedensity of cells cultured in a bioreactor disclosed herein. In someembodiments, a low flow rate is used to culture very high density cellcultures.

In some embodiments, bioreactor systems and bioreactors disclosed hereinare configured to also include one or more sensors suitably connected tothe controller for monitoring and/or regulating various physical and/orchemical parameters within the growth medium (such as, for example,temperature, pH, glucose concentration, dissolved oxygen concentrationthe concentration of dissolved carbon dioxide or of HCO₃ ⁻ ions, theconcentration of lactate, and ionic strength) in the growth medium, allcan be sensed monitored and controlled in the bioreactor and/orbioreactor headspace and/or in a fluid reservoir connectable to thebioreactor and/or at the various inlets or outlet ports. In someembodiments, sensors are configured to detect a product synthesized by acell or microorganism grown in the bioreactor. In some embodiments,control of some of the features above may require mixing of the growthmedium, the mixing can be provided at the fluid reservoir.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the bioreactors and systems thereof pertains.FIGS. 1-16 and the accompanying description thereof, provide numerousembodiments of bioreactors and systems thereof. A skilled artisan wouldrecognize that other methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments disclosed herein. In addition, the materials, methods, andexamples are illustrative only and are not intended to be necessarilylimiting.

Implementation of the method and/or system of embodiments of thebioreactor and systems thereof disclosed herein can involve performingor completing selected tasks manually, automatically, or a combinationthereof. Moreover, according to actual instrumentation and equipment ofembodiments of the method and/or system disclosed herein, severalselected tasks could be implemented by hardware, by software or byfirmware or by a combination thereof using an operating system.

For example, hardware for performing selected tasks according to someembodiments could be implemented as a chip or a circuit. As software,selected tasks according to some embodiments could be implemented as aplurality of software instructions being executed by a computer usingany suitable operating system.

In one embodiment, one or more tasks according to the methods and/orsystems as described herein, can be performed by a data processor, suchas a computing platform for executing a plurality of instructions.Optionally, the data processor includes a volatile memory for storinginstructions and/or data and/or a non-volatile storage, for example, amagnetic hard-disk and/or removable media, for storing instructionsand/or data. Optionally, a network connection is provided as well. Adisplay and/or a user input device such as a keyboard or mouse areoptionally provided as well.

Reference is now made to FIG. 1, which is a schematic partcross-sectional diagram illustrating a bioreactor system including abioreactor having a perforated barrier, in accordance with someembodiments of the bioreactors of the present application. According tosome embodiments, the bioreactor system 50 includes a bioreactor 10, apump 4, a controller 30 and a growth medium reservoir 20.

The pump 4 can be any type of fluid pump known in the art and capable ofreceiving a fluid such as a growth medium received at the pump's inletport and pumping it through an outlet port thereof at a controllablepumping rate without compromising the sterility of the growth medium.For example, the pump 4 can be a variable flow rate peristaltic pump,such as, for example, a model 530 process pump commercially availablefrom Watson-Marlow fluid technology group (UK) or any other suitabletype of pump known in the art.

The bioreactor 10 has a bioreactor wall 10A having a bottom part 10B anda top part 10C. In the embodiment of the bioreactor presented in FIG. 1,the bioreactor 10 comprises a top part 10C that has a threaded opening10E into which a threaded cover 10D is sealingly threaded. The cover 10Dis configured to also (optionally) have one or more openings thereinsuch as for example, the opening 10F into, which a sensor unit 22 isconfigured to be sealingly inserted into the volume enclosed within thewalls 10A of the bioreactor 10. According to some embodiments, thethreaded opening 10G is configured to be sealed by a threaded sealingcap 10H when not in use. The bioreactor cover 10D is configured to(optionally) include several additional sealable openings (not shown inFIG. 1), which are configured to be used for inserting thereinadditional sensors (not shown in FIG. 1), or other needed devices suchas, for example, a heating unit (not shown) an oxygenating unit (notshown), a thermometer (not shown) or any other device needed foroperating the bioreactor 10 and/or monitoring the contents of thebioreactor 10 and/or ports allowing sampling and introduction ofmaterials to the content of bioreactor 10.

According to some embodiments, the bioreactor 10 can be made from anysuitable biocompatible material known in the art, such as a suitablybiocompatible plastic or polymer based material. In some embodiments,the reactor 10 is made from a transparent material to enable an operatorto see the contents of the bioreactor 10. In some embodiments,non-limiting examples of materials that can be used in the constructionof the bioreactor 10 include but are not limited to, polystyrene,stainless steel, polyetheretherketone (PEEK), polysulfone, and varioustypes of polytetrafluoroethylene (PTFE) plastics, for example Rulon®. Insome embodiments, materials for use in the construction of a bioreactordescribed herein are selected based on their low coefficient offriction, excellent abrasion resistance, Gamma radiation sterilization,wide range of operating temperatures, or chemical inertness, or anycombination thereof.

The bioreactor 10 further comprises a perforated barrier 12 sealinglyattached to the walls 10A of the bioreactor 10. The perforated barrier12 divides the volume enclosed within the bioreactor 10 into a first(lower) chamber 14A and a second (upper) chamber 14B. The perforatedbarrier 12 is made from a material which has multiple perforationstherein. The average diameter of the perforations formed in theperforated barrier 12 is selected such that the cells 3 (ormicroorganisms) suspended in a growth medium 2 cannot penetrate into theperforations of the perforated barrier 12, while the growth medium 2 canflow into and through the perforations. The perforated barrier 12operates as a cell (or microorganism) barrier while allowing the growthmedium 2 to flow and pass there through. According to some embodimentsthe construction of the perforated barrier 12 is also configured toalign a medium flow. According to some embodiments, the alignmentcomprises improving the linearity and uniformity of a medium flowtowards the cell mass 3 and throughout the upper chamber.

The perforated barrier 12 can be made from any suitable perforatedbiocompatible material, such as, for example, a suitable biocompatibleplastic or polymer based material having a selected perforation averageperforation (or pore) diameter. The thickness and strength of theperforated barrier 12 and the type of perforated material selected forthe perforated barrier 12 can depend, for example, on the average sizeof the cells or microorganisms to be grown in the bioreactor 12, thedesired rate of flow of the growth medium 2 through the bioreactor, themaximal allowable level of pressure of the growth medium within thefirst chamber 14A, or the method of harvesting cells or microorganismsas implemented in the design of the bioreactor, or any combinationthereof. For example, if the perforated barrier needs to be flexible asexplained in detail hereinafter (See for example FIGS. 7-8), a thinnerperforated barrier can be selected for use. In some embodiments, thetypes of materials from which the perforated barriers can be made caninclude but are not limited to cellulose nitrate, cellulose acetate,polytetrafluorethylene (PTFE), hydrophobic PTFE, hydrophilic PTFE,aliphatic or semi-aromaticpolyamides—for example Nylon®, polycarbonate,polysulfone, polyethylene, polyethersoulfone, polyvinylidene, stainlesssteel, and regenerated cellulose.

In some embodiments, the thickness of the perforated barrier 12 can bein the range of 0.5-5.0 millimeter. In other embodiments, thinnerperforated barriers can be used depending on the application, themechanical properties of the material from which the perforated barrieris made, total surface area and shape of the perforated barrier andother considerations. In other embodiments, thicker perforated barrierscan be used depending on the application, the mechanical properties ofthe material from which the perforated barrier is made, total surfacearea and shape of the perforated barrier and other considerations.

The bioreactor 10 has a fluid inlet port 16 through which growth medium2 can be pumped into the first chamber 14A. The fluid inlet port 16 isconfigured to receive the growth medium 2 under pressure from the pump 4of the bioreactor system 50. The growth medium entering the fluid inletport 16 can pass into a fluid impeller 18 disposed within the firstchamber 14A. The (optional) fluid impeller 18 is configured to be ahollow disc-like perforated member having multiple passages 18P therein.

The fluid impeller 18 is configured to receive growth medium 2 from theinlet port 16 and disperse the growth medium 2 through the multipleperforations 18P in multiple jets 19 of growth medium to enhance themixing of the growth medium 2 entering the inlet port 16 with the growthmedium 2 disposed within the chamber 14A. It is noted that the specificstructure of fluid impeller 18 illustrated in FIG. 1 is one embodimentof a fluid impeller and not obligatory. Many other different types offluid impellers/dispersers having various different shapes, structures,dimensions and using passages and/or nozzles can be used, as is wellknown in the art including impeller types such as a pinch blade ormarine type.

According to some embodiments, in operation of the system 50, cells (ormicroorganisms) are suspended in a growth medium and placed within thesecond (upper) chamber 14B of the bioreactor 10 by inserting thesuspended cells through the opening 10E or through the opening 10G ofthe cover 10D (which can then be sealed with the cap 10H).Alternatively, the cell suspension can be inserted into the second(upper) chamber 14B through any other suitable port, such as forexample, a harvesting port 21 opening into the second chamber 14B justabove the surface 12A of the perforated barrier 12. The growth medium 2injected into the chamber 14B by the fluid impeller 18 increases thepressure of the growth medium 2 in the first chamber 14A and causes thegrowth medium 2 to flow through the perforations of the perforatedmember 12 into the second chamber 14B effectively perfusing the cellsmass 3 suspended in the growth medium 2 held within the second chamber14B. The growth medium 2 rises within the second chamber 14B and reachesthe level of a fluid outlet 26, where it is drained out of thebioreactor 10 and carried by a conduit 28 to the pump 4 where it isrecirculated into the bioreactor 12 through the inlet port 16.

In some embodiments, the bioreactor 10 has a generally frustoconicalshape. The diameter of the bottom part 10B is smaller than the diameterof the top part 10C and the walls 10A are sloped. Due to thefrustoconical shape of the bioreactor, the diameter of the bioreactorincreases as the growth medium moves upwards (towards the top part 10D)within the bioreactor.

As the pump 4 pushes the growth medium into the inlet port 16 at aconstant flow rate, the flow velocity (fluid velocity) of the growthmedium 2 adjacent the surface 12A of the perforated barrier 12 is higherthan the flow speed of the growth medium near the top part 10D,effectively resulting in establishing a fluid flow velocity gradientalong the longitudinal axis 35 of the bioreactor 10. The flow velocitygradient is schematically indicated by the length and thickness of thesolid arrows 37A, 37B and 37C. The flow velocity represented by thearrow 37A is greater than the flow velocity represented by the arrow 37Band the flow velocity represented by the arrow 37B is greater than theflow velocity represented by the arrow 37C.

The suspended cells 3 are carried upwards by the upward moving flow ofthe growth medium 2, which counteracts the tendency of the cells 3(which have a higher specific gravity than the specific gravity of thegrowth medium 2) to move downwards and to settle on the surface 12A dueto the force of gravity acting on the cells 3. The flow rate of thegrowth medium can therefore be controlled and adjusted to result in anadequate suspension of the cells within the volume of the growth medium2 contained in the second chamber 14B avoiding the settling of the cells3 on the surface 12A of the perforated barrier 12, while leaving most ofthe cells 3 suspended in the growth medium 2 at a region within thechamber 14B, which is adequately lower than the upper surface 2A of thegrowth medium 2 so as to minimize or adequately reduce the number ofcells entering the fluid outlet port 26 (which greatly reduces loss ofcells 3). According to some embodiments the outlet port 26 comprises aperforated barrier or filter (not shown), configured to prevent thecells or microorganisms from leaving the bioreactor. In someembodiments, the flow rate of the growth medium 2 through the secondchamber 14B is low enough to avoid substantial shear forces which can bedetrimental to the cells 3.

When the proper flow rate of the growth medium 2 through the bioreactor10 is established, the pump 4 circulates the growth medium 2 through thevolume of the bioreactor 10 by pumping any growth medium 2 exiting thefluid outlet port 26 back into the bioreactor through the fluid inletport 16 in a closed loop. During the cell growth, when there arises aneed to add new nutrients to the growth medium 2 (to compensate fordepletion thereof by absorption into cells) or to add activatingsubstances or any other additive or substance into the growth medium 2,this can be done by flowing some fresh growth medium 2 from the mediumreservoir 20 of the system 50 by way of a media tube (38).

The medium reservoir 20 is configured to be connected to an inlet port4A of the pump 4 by a suitable hollow conduit 38. A suitablecontrollable valve (or stopcock) 39 is configured to be attached betweenthe conduit 38 and the pump inlet 4A, such that the flow of growthmedium from the fluid reservoir 20 into the pump inlet port 4A can becontrolled. The valve 39 is configured to be controllably closed to stopfeeding fluid from fluid reservoir 20 into the pump inlet port 4A or isconfigured to be opened to enable feeding fluid from fluid reservoir 20into the pump inlet port 4A allowing media refreshment and high densitycell culturing.

In some embodiments, regulation of flow rates correlates with thedensity of cells being grown and propagated. In some embodiments, verylow flow rates provide for high density culturing of cells in thebioreactors disclosed herein. In some embodiments, the working volume ofmedia in which the cells are grown is low, as is the flow rate allowingfor the maintenance of high density culturing of cells. This low workingvolume and low flow rate, can in certain embodiments, lead to higheryields and lower media needs. In some embodiments, the bioreactorsdisclosed herein and methods of use thereof, are advantageous comparedwith other bioreactors known in the art due to their ability achieve andmaintain high density cultures of cells or organisms, which results inhigher yield and lower media needs. In some embodiments, a bioreactordisclosed herein comprises a smaller physical footprint minimizing thebioreactor size, and thereby reducing media use.

According to some embodiments, the bioreactor 10 is configured to also(optionally) include an additional outlet port 27 opening at the bottompart 10B of the bioreactor. The outlet port 27 includes a valve (orstopcock) 25 that is configured to allow draining an amount of thegrowth medium 2 from the first chamber 14A of the bioreactor 10 ifnecessary. For example, if an amount of new growth medium 2 is added tothe bioreactor 10 from the fluid reservoir 20, a similar amount ofgrowth medium can be bled out of the bioreactor 10 to restore the levelof growth medium 2 within the second chamber 14B.

According to some embodiments, growth medium 2 can also be bled out ofthe bioreactor 10 through the outlet port 25 when it is desired toreduce the total volume of the growth medium 2 within the second chamber14B in order to concentrate the cells 3 for cell harvesting. When such acell concentrating is performed, the smaller volume of the growth medium2 remaining in the lower part of the second chamber 14B has a highercell count (in cells/ml of growth medium) since the cells 3 cannot passthe perforated barrier 12 and are therefore concentrating. Theconcentrated suspension of cells 3 remaining in the chamber 14B can thenbe harvested through the harvesting port 21 which is configured toinclude a valve (or stopcock) 23 as illustrated in FIG. 1.

In some embodiments, in order to prevent clogging the perforated barrier12 most the growth medium can be drained via at least one of the outletports 126A-126D (detailed in the following), and only a minimal volumeof the growth medium may be drained via outlet port 25.

It is noted that while any desired additives and/or substances can beintroduced into the bioreactor 10 by introducing such substances and/oradditives into the growth medium 2 held within the fluid reservoir 20and allowing a volume of the growth medium 2 including such substancesand/or additives to flow into the chamber 14A, as disclosed hereinabove,it can also be possible to directly introduce such substances and/oradditives into the bioreactor by introducing a relatively small volumeof fluid or growth medium including a suitably high concentration of thesubstances and/or additives into the bioreactor 10 through any suitableopening or inlet port of the bioreactor 10 and allowing the added smallvolume to mix with the volume of growth medium 2 circulating within thereactor to reach the desired concentration. For example, such smallvolumes of fluid or growth medium including additives and/or substancescan be introduced through the opening 10G by temporarily removing thecap 10H and resealing the opening 10G.

In some other embodiments, the cap 10H is configured to include apenetrable sealing diaphragm (not shown in detail in FIG. 1) made fromrubber, latex or any other suitable sealing material as is known in theart and commonly used in bottles containing injectable liquidformulations and the small volume of fluid with substances and/oradditives can be loaded within a sterile syringe having a sterilizedneedle and where the needle is configured to be pushed into the sealingdiaphragm of the cap until it penetrates the sealing diaphragm, thecontents of the syringe can then be injected into the growth medium 2within the second chamber 14B, and the needle of the injector can bewithdrawn from the sealing membrane as is known in the art. This methodcan advantageously reduce the risk of contamination of the growth mediumby any undesirable microorganisms. Additionally, cap 10H is configuredto have a deep tube touching the growth medium 2 with a one way sealallowing media sampling in a sterile way.

In some other embodiments, the cap 10H is configured to include a filter(not shown in FIG. 1). The cap's filter is configured to allow a flow ofair to the headspace (space between the bioreactor top 10C and themedia's surface) or for reduction pressure from the headspace.

According to some embodiments, the bioreactor system 50 is configured touse the controller 30 and the sensor unit 22 for monitoring theoperation of the system. The sensor unit 22 is configured to include asensor or multiple sensors (the individual sensors are not shown indetail in FIG. 1 for the sake of clarity of illustration), which can bedisposed in several locations for example: via the end part 22A of thesensor unit 22 that is immersed in the growth medium 2, or via at leastone of the outlet ports (126A-126D), or via harvesting port (21), or viainlet port (116), or via outlet port (27) or via side wall (10A) or anycombination thereof. The sensor(s) of the sensor unit 22 can be used todetermine the concentration of several chemical species within thegrowth medium 2, such as, for example, the concentration of H⁺ ions (todetermine the pH of the growth medium 2), the concentration of dissolvedoxygen in the growth medium 2, the concentration of dissolved carbondioxide in the growth medium 2 or of HCO₃ ⁻ ions in the growth medium 2,the concentration of glucose, the concentration of lactate, and ionicstrength. Such sensor or sensors can be single use sensors using opticsensing without the need to penetrate the wall or can be located on 10Atouching the liquid. According to some embodiments, the sensors of thesensor unit 22 are configured to also be sensors for sensing physicalparameters of the growth medium 2, such as but not limited to, thetemperature and/or the turbidity and/or the optical density of thegrowth medium 2, and/or any other desired physical parameter of thegrowth medium 2 such as, conductivity, capacitance, pressure, flowrates, viscosity, turbidity and others.

According to some embodiments, the signal(s) from the sensor unit 22representing any of the chemical and/or physical parameters sensed bythe sensors can be fed into the controller 30 by suitable electricalconductors (or conductor pairs) 22B. The controller 30 is configured toprocess such sensor signals to determine of the values of the sensedparameters as is well known in the art.

According to some embodiments, the controller 30 is configured to be orconfigured to include one or more processing devices such as, forexample, a microprocessor or a microcontroller or a digital signalprocessor, a personal computer or any other suitable means forprocessing received signals and any type of memory device known in theart for storing any computed data therein for the purpose of off-line oron-line presentation of all determined sensor data and the history ofoperation of the bioreactor (including, but not limited to, the rate offlow of growth medium 2 through the bioreactor 10, the time ofintroducing and the volume of growth medium from the fluid reservoir 20,the time of introducing and the volume and concentration of any otheradded substance or additive during the operation of the system 50).

According to some embodiments, the controller 30 is configured to alsoinclude any display device known in the art for displaying processedresults and the values of any sensed parameters to an operator or userof the system 50. The controller 30 is configured to also include one ormore user interface device (such as, but not limited to a mouse, a lightpen, a pointing device, a keyboard, a touch sensitive screen, or anyother input device known in the art) which is configured to be used bythe user or operator of the system 50 for inputting data and/or suitablecommands into the controller 30. For example, the user can control therate of flow of the growth medium 2 through the bioreactor 10 byentering suitable commands into the controller 30 resulting in suitablecontrol signals being sent by the controller 30 to the pump 4 through acommunication line 29 connecting the controller 30 and the pump 4.

In some embodiments of the systems of the present application, thevalves 23, 24, 25, and 39 of the system 50 are configured to be manualvalves or stopcocks, which can be manually closed or opened. In someother embodiments, one or more of the valves 23, 24, 25, and 39 areconfigured to be electrically operated valves that can be operated byreceiving appropriate command signals from the controller 30.

For example, any of the valves 23, 24, 25, and 39 can be electricallyoperable solenoid based valves which can be opened and/or closedcontrollably and/or automatically by applying suitable voltage orcurrent signals to the solenoids by the controller 30. It is noted thatfor the sake of clarity of illustration any electrical wires connectedbetween the controller 30 and any of the valves 23, 24, 25, and 39 arenot shown in FIG. 1. However, such optional connections are shown in theschematic diagram of FIG. 5.

It is noted that while in the bioreactor system 50 the level of theupper surface 2A of growth medium 2 in the second chamber 14B is fixed,this is not obligatory and in some embodiments of the bioreactorsystems, the level (height) of the growth medium in the bioreactor canbe controllably changed.

Reference is now made to FIG. 2, which is a schematic partcross-sectional diagram illustrating a bioreactor system having abioreactor with multiple fluid outlet ports for controllably adjustingthe level of the growth medium in the bioreactor, in accordance withsome embodiments of the bioreactors of the present application.

According to some embodiments, the bioreactor system 150 includes abioreactor 110, the controller 30 as disclosed in detail hereinabove,the pump 4 as disclosed in detail hereinabove and the fluid reservoir 20as disclosed in detail hereinabove. The bioreactor system 150 isconfigured to also include an oxygenating system 160. The bioreactor 110can be made from any of the materials disclosed in detail hereinabovefor the bioreactor 110. The bioreactor 110 has a bioreactor wall 110A, abottom part 110B and a bioreactor top part 110C. According to someembodiments, the top part 110C is configured to have a threaded opening110F therein for sealingly inserting there through a threaded sensorunit 122. A top opening in the top of the bioreactor 110D can beeffectively closed using a cap 110E, wherein the seal of the opening inthe head plate of the bioreactor is represented by 110G.

According to some embodiments, the sensor unit 122 is configured toinclude any number of sensors (not shown individually in FIG. 2 for thesake of clarity of illustration) attached to or included in the end 122Aof the sensor unit 122 for sensing any desired chemical or physicalproperty of the growth medium 2 within, which the end 122A of the sensorunit 122 can be immersed. It is noted that the position of the end 122Acan be changed by threading the sensor unit 122 up or down within thethreaded opening 110F such that the end 122A can be immersed in thegrowth medium 2 at any level of the growth medium 2 within thebioreactor 110.

A perforated barrier 112 is sealingly attached to the wall 110A of thebioreactor 110 such that the perforated barrier 112 divides the internalvolume of the bioreactor 110 into a first (lower) chamber 114A and asecond (upper) chamber 114B, as disclosed in detail hereinabove for thebioreactor 10 and the perforated barrier 12 of the bioreactor system150. According to some embodiments, the perforated barrier 112 can bemade from similar material(s) and can have similar perforation meansizes as disclosed in detail hereinabove for the perforated barrier 12.

However, according to some embodiments, while the bioreactor 10 (ofFIG. 1) has a single fluid outlet port 26 in the second chamber 14B, thebioreactor 110 has plurality of different fluid outlet ports atdifferent heights and corresponding valves, for example four differentfluid outlet ports 126A, 126B, 126C and 126D in the second chamber 114B.The outlet ports 126A, 126B, 126C and 126D are disposed along the lengthof the second chamber 114B at different positions and each of the fluidoutlet ports outlet ports 126A, 126B, 126C and 126D has a correspondingvalve 124A, 124B, 124C and 124D (respectively) attached thereto. Thevalves 124A, 124B, 124C and 124D are fluidically connected to a commonfluid manifold 128 which is fluidically connected to the pump 4. Thearrangement of the four valves 124A, 124B, 124C and 124D at differentpositions allows the level of the growth medium 2 to be selected fromfour different levels schematically represented in FIG. 2 by the dashedlines A, B, and C and the line D.

In some embodiments, if the valve 124D is opened and the valves 124A,124B, 124C are closed (as illustrated in FIG. 2), the growth medium 2reaches the level represented by the solid line D and the growth medium2 leaving the second chamber 114B through the fluid outlet port 126Denters the manifold 128 and is re-circulated into the bioreactor 110 bythe pump 4 pumping the growth medium 2 through the pump outlet 4B intothe fluid inlet port 116 and through the perforations 19 the fluidimpeller 18.

In some embodiments, if it is desired to increase the level of growthmedium 2 in the second chamber 114B, the valves 126A, 126B and 126D canbe closed and the valve 126C can be opened while the valve 39 can beopened for a period of time allowing an amount of growth medium 2 fromthe reservoir 20 to be pumped by the pump 4 into the first chamber 114Auntil the level of the growth medium 2 to reach the level represented bythe dashed line C at which time the valve 39 can be closed and thegrowth medium 2 leaves the second chamber through the fluid outlet port126C.

Similarly, in some embodiments if it is desired to further increase thelevel of growth medium 2 in the second chamber 114B, the valves 126A,126C and 126D can be closed and the valve 126B can be opened while thevalve 39 can be opened for a period of time allowing an additionalamount of growth medium 2 from the reservoir 20 to be pumped by the pump4 into the first chamber 114A until the level of the growth medium 2 toreach the level represented by the dashed line B at which time the valve39 can be closed and the growth medium 2 leaves the second chamberthrough the fluid outlet port 126B.

Furthermore, if it is desired to even further increase the level ofgrowth medium in the second chamber 114B, the valves, 126B, 126C and126D, according to some embodiments, can be closed and the valve 126Aopened while the valve 39 can be opened for a period of time allowing anadditional amount of growth medium 2 from the reservoir 20 to be pumpedby the pump 4 into the first chamber 114A until the level of the growthmedium 2 reaches the level represented by the dashed line A, at whichtime the valve 39 can be closed and the growth medium 2 leaves thesecond chamber through the fluid outlet port 126A.

It will be appreciated by those skilled in the art that while thebioreactor 110 includes four fluid outlet ports 126A, 126B, 126C and126D levels allowing four different levels, this is not obligatory ofthe growth medium 2 to be achieved during closed loop perfusion(recirculation) of the growth medium 2, this is by no means intended tobe obligatory. Rather, in some embodiments of the bioreactors of thepresent applications, the number of the outlet ports (and thecorresponding valves attached thereto) opening into the second chamberof the bioreactor can be varied as desired and can be smaller or largerthan four (with suitable modification of the manifold 128 to accommodatethe required number of valves), in such a way as to allow any desiredpractical number of growth medium 2 levels to be achieved in the secondchamber of the bioreactor by suitable opening and closing of the valvesas disclosed in detail hereinabove.

An advantage of being able to set different levels of growth medium 2within the second chamber of the bioreactor is that it can allow theincreasing or decreasing of the total volume of growth medium 2 in thesecond chamber 114B in order to increase (or decrease, respectively) thenumber of cells (or microorganisms) which can be grown within thebioreactor, if necessary. This mechanism allows culturing of cells inhigh density and adapting the refreshment of media and nutrients as thecell proliferate reducing or eliminating the need for passaging anddish/container replacement.

According to some embodiments, at least some of the plurality ofdifferent fluid outlet ports at the different heights and together withtheir corresponding valves are configured also as fluid inlets ports. Insome embodiments, the plurality of different fluid outlet/inlet ports isconfigured to circulate out of the bioreactor a portion of the cells ormicroorganisms. In some embodiments, cells or microorganisms may becirculated out of the upper chamber of the bioreactor in order toprocess cells wherein the processed cells are then circulated back intothe bioreactor (not shown). In some embodiments, cells may for examplebe selected by depleting or enriching of a specific cell type orgenetically modified, for example but not limited to, to express apolypeptide or fragment thereof not previously expressed, or to increaseor decrease expression of a polypeptide or fragment thereof. In someembodiments, processing comprising inducing cells to increase ordecrease expression of a specific gene or gene variant. Methods ofgenetic modification and control of gene expression are well known inthe art. In some embodiments, cells may be transformed (geneticallymodified) using any method known in the art. In some embodiments, cellsmay be processed wherein polypeptide expression is modified using anymethod known in the art. In a related embodiment, the outlet/inlet fluidports and their corresponding valves are selected to circulate the cellmass, according to the cells mass current level (height).

It is noted that according to some embodiments, the frustoconical shapeof the bioreactor 110 allows the establishment of a fluid velocitygradient along the length of the bioreactor 110 in order to gently floatthe cells mass 3 and keep most of the cells mass 3 suspended within adefined region of the growth medium 2 contained in the second chamber114B to avoid cell accumulation on (and/or adhering) to the uppersurface 112A of the perforated barrier 112 as well as to reduce cellloss by exiting through a fluid outlet port being used for recirculationof the growth medium 2.

According to some embodiments, the provided bioreactor comprises avessel or at least an upper chamber with an inverted frustoconical shapeconfigured to allow the cell (or microorganism) growing mass to floatand elevate to a larger surface, due to the medium's upstream flow(against gravity direction) and the pressure equilibrium (mass gravityvs. upstream liquid's flow). Further, due to constant volumetric-flow, aslower flow of the medium runs through the cell (or microorganism) massat the upper and larger areas of the inverted frustoconical shape, whichassist in concentrating the cells mass, and reduces shear forces appliedby the medium's flow.

It is noted that like in the bioreactor 10 of FIG. 1, the vessel walls110A are slanted at an angle with respect to a longitudinal axis 135 ofthe bioreactor 110 as can be seen in the part longitudinal cross sectionview of FIG. 2. According to some embodiments, the angle at which thevessel walls 110A are configured to be slanted with respect to thelongitudinal axis 135 can be in the range of 0 to 175 degrees. However,higher or lower slant angles can also be used, depending, inter alia, onthe particular application. It is noted that not all the walls of thebioreactors of the present application need be slanted and only some ofthe walls are configured to be slanted depending on the specific shapeof the bioreactor (for example, see the bioreactor of FIG. 4I, hereinafter). Thus, the area of a transversal cross section of the bioreactortaken at a level represented by the dashed line A is larger than thearea of a transversal cross section taken at a level D.

According to some embodiments, the transversal cross sectional area ofthe bioreactor 110 becomes larger as one moves upwards along thelongitudinal axis 135 within the second chamber 114B results in theestablishing of a fluid velocity gradient in the growth medium 2 suchthat the fluid velocity of the growth medium 2 gradually decreases asone moves upwards in the direction from the surface 112A towards the toppart 110C.

This fluid velocity gradient assists in suspending most of the cells ormicroorganisms in a zone or region within the growth medium 2 of thesecond chamber 114B in which the force of gravity acting downwards onthe cells 3 (or microorganisms) balances out the mean upward directedforce exerted on the cells by the upward flowing growth medium 2 as isdisclosed in detail hereinabove for the bioreactor 10. Thus, in thebioreactor 110, the controlling of the level (or height) of the growthmedium 2 within the second chamber 114B together with controlling of theflow rate of the growth medium 2 (by controlling the pump flow rate) canadvantageously allow finer control of the zone or region within whichmost of the cells are suspended within the second chamber 2.Additionally, the flow rate control allows minimizing the sheer forcesintroduced to the cells and maintains the ability to optimize andrefresh media in correlation to the cells proliferation and densitywhich could result in high cell density culturing.

According to some embodiments, the perforated barrier 112 of thebioreactor 110 is a flat (planar) barrier. According to someembodiments, a harvesting port 127 is configured to be used forharvesting cells from the bioreactor 110. According to some embodiments,the harvesting port 127 is shaped as a hollow member or tube thatincludes a first hollow part 127A and a second hollow part 127B. Thepart 127A is sealingly attached to the perforated barrier 112 (in someembodiments at the center of the perforated barrier 112) and has anopening 127C which opens into the second chamber 114B at the uppersurface 112A of the perforated barrier.

The second hollow part 127B is contiguous with the first hollow member127A and bent at an angle thereto such that it passes through the vesselwall 110A of the first chamber 114A and is sealingly attached to thevessel walls 110A. The second part 127B exits the vessel walls 110A andextends outside the bioreactor 110. The second part 127B includes avalve (or a stopcock) 123 which is disposed within the portion of thesecond part 127B that extends outside of the bioreactor 110. When it isdesired to harvest cells 3 from the bioreactor, this can be performed byconcentrating the cells by reducing the level of the growth medium 2within the second chamber 114B.

For example, the level of the growth medium 2 can be brought to thelevel represented by the line D, or, alternatively, to a level lowerthan the level D by draining additional growth medium from the firstchamber through a suitable outlet port (not shown in FIG. 2) disposed inthe bottom part 110B of the bioreactor 110 (such as, for example, anoutlet port similar to the outlet port 27 or ports 126A-126D illustratedin FIG. 1). After the cells 3 are concentrated, the suspension of cells3 in the growth medium 2 can be harvested through the harvesting port127 by opening the valve 123 and receiving the cell suspension in anappropriate collecting vessel (not shown).

According to some embodiments, the valves 126A, 126B, 126C, 126D, 39 and123 can be manual valves (or stopcocks), but may, in accordance withsome embodiments of the bioreactor 110, controllably and/orautomatically operable as disclosed in detail hereinabove with respectto the valves 24, 23, 25 and 39 of FIG. 1. For example, any of thevalves 126A, 126B, 126C, 126D, 39 and 123 are configured to beelectrically operable solenoid valves which can be controlled to openand closed by the controller 30 of the bioreactor system 150 (it isnoted that any lines connecting any of the valves 126A, 126B, 126C,126D, 39 and 123 to the controller 30 if the valves are indeedimplemented as solenoid based valves, are not shown in FIG. 2 for thesake of clarity of illustration. However, such schematic lines are shownin more detail in FIG. 5 hereinafter). According to some embodiments,the controller 20 is configured to be suitably connected throughconnecting wires 22B to a sensor unit 122 which is configured to includeany number of sensors for sensing any chemical and/or physicalproperties of the growth medium 2 as disclosed in detail hereinabove forthe sensor unit 22 of FIG. 1. It is noted that while the position of theend 22A of the sensor unit 22 can be fixed (since the level of thegrowth medium 2 in the second chamber 14B of the bioreactor 10 does notchange significantly during perfusion, the sensor unit 122 is configuredto be substantially longer than the sensor unit 22 and is configured tobe implemented in such a way that the position of the end 122A of thesensor unit 122 can be changed, if necessary to accommodate any changesin the level of the surface of the growth medium 2 within the secondchamber 114B.

For example, a substantial part of the length of the sensor unit 122 canbe threaded and the opening 110F, into which the sensor unit 122 fits,can also be internally threaded to allow changing the position of theend 122A within the second chamber by suitably screwing the sensor 122in or out as necessary. Alternatively, the surface of the sensor unit122 can be smooth and the position of the end 122A of the sensor 122 canbe varied by suitably sealingly pushing or pulling the sensor unit 122within a suitable gasket (not shown in FIG. 2) sealingly disposedbetween the opening 110F and the sensor unit 122.

According to some embodiments, the oxygenating system 160 of the system150 is configured to include an oxygen source 160A for supplying oxygengas to the bioreactor 110, and a gas dispersing head 160 (optionally)disposed within the first chamber 114A. According to some embodiments,the oxygen source 160A is configured to be connected through a gas valve160D to the gas dispersing head by a suitable hollow member 160Csealingly passing through the wall 110A of the bioreactor 110 such as,for example Suitable hollow flexible tubing. Alternatively, according tosome embodiments, the oxygen source 160A is configured to be suitablyconnected through a suitable gas valve 160D to a fixed inlet formed asan integral part of the wall 110A to which the gas dispersing head canbe suitably attached.

According to some embodiments, the gas valve 160D is configured to be amanually operated valve manually opened or closed by an operator.However, in some embodiments, the gas valve 160D may is configured to bean actuator controlled valve that can be suitably opened or closed byreceiving suitable electrical command signals from the controller 30 (itis noted that any command lines connecting the controller 30 with thegas valve 160D are not shown in FIG. 2 for the sake of clarity ofillustration. According to some embodiments, the oxygen source 160A canbe a compressed oxygen tank as is known in the art, but canalternatively be any type of oxygen generator known in the art, such asbut not limited to an electrolytic oxygen generator or any other sourceof gaseous oxygen known in the art. Alternatively, the oxygen source canbe a source of any mixture of gases which contains a substantial amountof oxygen (such as, for example, air, a mixture of oxygen and nitrogen,a mixture of oxygen, nitrogen and carbon dioxide, or any other suitablemixture of gases suitable for the purpose of oxygenation of a growthmedium as is known in the art.). According to some other embodiments,the oxygenation of the liquid medium is provided at the liquid'sreservoir 20.

When the gas valve 160D is open, oxygen gas from the oxygen source 160Apasses through the gas dispersing head 160B and is dispersed in the formof small oxygen containing bubbles that rise up within the first chamber114A. The gas dispersing head 160B can be any type of head includingperforations therein and capable of dispersing a gas passing therethrough a liquid (such as, for example the growth medium 2) in the formof small bubbles. For example, the gas dispersing head 160B can be ablock of perforated ceramic material, a block of perforated stainlesssteel, a block of perforated titanium, or any other type of sterilizabledispersing head known in the art (such a gas dispersing head can besimilar in construction and operation to the gas dispersing heads usedto oxygenate the water in fish aquaria, as is well known in the art).

It is noted that while the oxygenating system 160 illustrated in FIG. 2directly provides oxygen to the growth medium within the first (lower)chamber 114A of the bioreactor 110, this is in no way obligatory forpracticing the bioreactor or bioreactor systems disclosed herein. Forexample, the oxygenating system 160 can provide oxygen to otherdifferent parts of the bioreactor system 150, such as, for example tothe second chamber 114B or to the manifold 128, or to the fluidreservoir 20, or can provide oxygen to more than one part of thebioreactor system 150 (such as, for example, both to the first chamber114A and to the fluid reservoir 20).

Alternatively, the oxygen level in the medium can be controlled bycontrolling the oxygen levels in the headspace between the bioreactortop 110C and the media D surface allowing oxidation by diffusion. Thiscan be implemented by placing the oxygen dispersing head 160B in thedesired part of the system or by providing several oxygen dispersingheads all suitable connected to the oxygen source 160A and disposed inany selected parts of the bioreactor system 150 for oxygenating anygrowth medium disposed in such parts. All such alternative oxygen supplymethods are contemplated for use in some of the embodiments of thebioreactors and/or bioreactor systems as disclosed herein.

It is further noted that, since the sensors, for example the dissolvedoxygen sensor, can be placed in the various inlets and outlets of thebioreactor (as mentioned above), the monitoring of the dissolved oxygenconcentration within the growth medium is enabled at any time or processstage (either continuously, or at preset and/or programmable and/orpredetermined time intervals). Accordingly, it enables to automate theoxygenation of the growth medium 2 in the bioreactor 110 byautomatically regulating the rate of gas flow of oxygen (or oxygencontaining gas mixture) through the dispersing head 160B (or heads ifthere is more than one such head in the system 150) to maintain adesired level of dissolved oxygen in the growth medium. According tosome embodiments, the increasing of the medium's oxygen level, at thebioreactor vessel, can be provided by increasing the medium's oxygenlevel at the reservoir, and by increasing perfusion rate of the mediumat the first chamber.

It is noted that the shape of the bioreactors of the present applicationare not limited to the frustoconical shape as illustrated in FIGS. 1-2.For example, the bioreactors are configured to have, inter alia, conicalshape, a frustoconical shape, a tapering shape, a cylindrical shape, apolygonal prism shape, a tapering shape having an ellipsoidaltransversal cross section, a tapering shape having a polygonaltransversal cross section, a shape having a cylindrical part and atapering part, and a shape having a conical or tapered part and ahemispherical part. However, other different bioreactor shapes can alsobe implemented in accordance with some embodiments of the bioreactor,depending, inter alia, on the specific application and on manufacturingconsiderations.

Several possible exemplary shapes of the bioreactors are schematicallyillustrated in FIG. 3 and FIGS. 4A-4I. Reference is now made to FIG. 3,which is a schematic part cross-sectional diagram illustrating abioreactor system including a bioreactor having a cylindrical shapeincluding a perforated barrier, in accordance with another embodiment ofthe bioreactors of the present application.

According to some embodiments, the bioreactor system 250 includes abioreactor 210, the controller 30 as disclosed in detail hereinabove,the pump 4 as disclosed in detail hereinabove and the fluid reservoir 20as disclosed in detail hereinabove. According to some embodiments, thebioreactor system 250 also includes the oxygenating system 160 asdisclosed in detail hereinabove. The bioreactor 210 can be made from anyof the materials disclosed in detail hereinabove for the bioreactors 10and 110. The bioreactor 210 has vessel walls 210A, a bottom part 210Band a bioreactor top part 210C. The top part 210C may have an opening210G therein and a self-sealing gasket 211 can be disposed within theopening for sealing the opening. The self-sealing gasket 211 can besealably penetrated by a needle (not shown in FIG. 3) for introducing asuspension of cells or microorganisms in a growth medium, or any otherfluid or solution containing any substance or additive into thebioreactor 210, as disclosed in detail hereinabove.

It is noted that the cells or microorganisms can also be introduced intothe second chamber of the bioreactor through any suitable one way valve(not shown in FIG. 3) disposed in the walls or top of the bioreactorsuch that the one way valve allows the injecting of a cell suspension ora microorganism suspension there through and into the second chamber ofthe bioreactor without compromising the sterility of the bioreactor.

In accordance with one embodiment of the bioreactors, the one way valvecan be a luer-lock like valve which can be shaped to accept the end of astandard syringe containing the cell or microorganism suspension. Theuse of such a one way valve can be advantageous because the orifice ofthe valve can be made sufficiently large to reduce the shearing forcesaffecting the cells when the suspension is injected into the bioreactor.It is noted that any of the bioreactors of the present application areconfigured to have any combination of such opening(s), self-sealinggasket(s) and one way valve(s).

According to some embodiments, the vessel walls 210A are configured tohave an opening 210F for sealingly inserting there through a threadedsensor unit 222. The sensor unit 222 is configured to include any numberof sensors (not shown individually in FIG. 3 for the sake of clarity ofillustration) attached to or included in sensor unit 222 for sensing anydesired chemical or physical property of the growth medium 2 asdisclosed in detail hereinabove with respect to the sensor unit 122 inFIG. 2.

According to some embodiments, a perforated barrier 212 is sealinglyattached to the vessel wall 210A of the bioreactor 210 such that theperforated barrier 212 divides the internal volume of the bioreactor 210into a first (lower) chamber 214A and a second (upper) chamber 214B, asdisclosed in detail hereinabove for the bioreactor 10 and the perforatedbarrier 12 of the bioreactor system 50 of FIG. 1. The perforated barrier212 can be made from similar material(s) and can have similarperforation mean sizes as disclosed in detail hereinabove for theperforated barriers, for example 12 of FIG. 1. However, while thebioreactor 10 (of FIG. 1) has a single fluid outlet port 26 in thesecond chamber 14B, the bioreactor 210 can comprise several differentfluid outlet ports (not shown) in the second chamber 214B, wherein theoutlet ports comprise an individual outlet and valve (not shown).

According to some embodiments, the valves are fluidically connected to acommon fluid manifold 280A which is fluidically connected to the pump 4.The arrangement of the four valves at different positions, asillustrated in FIG. 2, allows the level of the growth medium 2 to beselected from four different levels.

According to some embodiments, the number of the outlet ports (and thecorresponding valves attached thereto) opening into the second chamberof the bioreactor can be varied (the number of outlet ports can besmaller or larger than 4, with suitable modification of the manifold 280to accommodate the required number of valves) in such a way as to allowany desired practical number of growth medium 2 levels to be achieved inthe second chamber of the bioreactor by suitable opening and closing ofthe valves as disclosed in detail hereinabove.

According to some embodiments, the oxygenating system 160 of the system250 includes an oxygen source 160A for supplying oxygen gas to thebioreactor 110, and a gas dispersing head 160 (optionally) disposedwithin the first chamber 214A. The oxygen source 160A is configured tobe connected through a gas valve 160D to the gas dispersing head by asuitable hollow member 160C sealingly passing through the wall 210A ofthe bioreactor 110 such as, for example Suitable hollow flexible tubing.Alternatively, the oxygen source 160A is configured to be suitablyconnected through a suitable gas valve 160D to a fixed inlet formed asan integral part of the wall 210A to which the gas dispersing head canbe suitably attached. Additionally, the concentration of oxygen can alsobe controlled by controlling the oxygen concentration in the headspacebetween the top part 210C and liquid level D allowing oxygenation of thegrowth medium 2 via diffusion. In some embodiments, the pH may beadjusted. For example but not limited to controlling CO₂ concentration,the pH can be controlled by controlling the CO₂ concentration in theheadspace via diffusion.

According to some embodiments, the gas valve 160D is configured to be amanually operated valve manually opened or closed by an operator.However, in some embodiments, the gas valve 160D is configured to be anactuator controlled valve that can be suitably opened or closed byreceiving suitable electrical command signals from the controller 30 (itis noted that any command lines connecting the controller 30 with thegas valve 160D are not shown in FIG. 3 for the sake of clarity ofillustration. According to some embodiments, the oxygen source 160A canbe a compressed oxygen tank, as is known in the art, but canalternatively be any type of oxygen generator known in the art, such asbut not limited to an electrolytic oxygen generator or any other sourceof gaseous oxygen known in the art.

Alternatively, the oxygen source can be a source of any mixture of gaseswhich contains a substantial amount of oxygen (such as, for example,air, a mixture of oxygen and nitrogen, a mixture of oxygen, nitrogen andcarbon dioxide, or any other suitable mixture of gases suitable for thepurpose of oxygenation of a growth medium as is known in the art.) Whenthe gas valve 160D is open, oxygen gas from the oxygen source 160Apasses through the gas dispersing head 160B and is dispersed in the formof small oxygen containing bubbles that rise up within the first chamber214A. The gas dispersing head 160B can be any type of head includingperforations therein and capable of dispersing a gas passing through aliquid (such as, for example the growth medium 2) in the form of smallbubbles.

For example, the gas dispersing head 160B can be a block of perforatedceramic material, a block of perforated stainless steel, a block ofperforated titanium, or any other type of sterilizable dispersing headknown in the art (such a gas dispersing head can be similar inconstruction and operation to the gas dispersing heads used to oxygenatethe water in fish aquaria, as is well known in the art).

Reference is now made to FIGS. 4A-4I which are schematic cross-sectionaldiagrams illustrating several exemplary shapes of bioreactors includinga perforated barrier in accordance with several embodiments of thebioreactors of the present application. It is noted that, for the sakeof clarity of illustration, the schematic drawings of FIGS. 4A-4Iillustrate only the general shape of the walls of the bioreactors andthe perforated barrier included therein and do not show any details ofany additional components of the bioreactors or bioreactor systems (suchas, for example, various openings in the walls of the bioreactors,sensor units, fluid inlet ports, fluid outlet ports, draining ports,harvesting ports, heating units, cooling/heating units, fluid impellers,gas dispersing heads, valves, pumps, controllers, self-sealable gaskets,fluid manifolds or any other components) which are not important tounderstanding the shape of the bioreactors. It will be appreciated bythose skilled in the art that any such components not shown in FIGS.4A-4I may be included in any non mutually exclusive combinations and/orpermutations in any of the bioreactors schematically illustrated inFIGS. 4A-4I, as is disclosed herein in detail herein and illustrated inthe drawing figures.

It is further noted that while the perforated barriers illustrated inFIGS. 4A-4I are illustrated as a flat fixed perforated barriers, this isshown by way of example only and it is contemplated that any of thebioreactors having shapes as disclosed in FIGS. 4A-4I may also beimplemented as any of the types of perforated barriers disclosed in thepresent application (including any of the flat or non-flat, fixed andmovable perforated barriers, buckling perforated barriers and all otherperforated barrier forms disclosed in the present application).

Turning to FIG. 4A, the bioreactor 300 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 300 into afirst chamber 304A shaped as a cylindrical part of the bioreactor 300and a second chamber 304B shaped as a frustoconical part of thebioreactor 300. Thus the bioreactor 300 has a shape that has acylindrical part and a frustoconical part.

Turning to FIG. 4B, the bioreactor 310 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 310 into afirst chamber 314A shaped as a cylindrical part of the bioreactor 300and a second chamber 314B shaped as a tapering part of the bioreactor300. Thus, the bioreactor 300 has a shape that has a cylindrical partand a tapering part. The tapering walls 308 of the second chamber 314Bhave a convex outer surface 308A.

Turning to FIG. 4C, the bioreactor 320 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 320 into afirst chamber 324A shaped as a cylindrical part of the bioreactor 320and a second chamber 324B shaped as a tapering part of the bioreactor320. The bioreactor 320 has a shape that has a cylindrical part and atapering part. The tapering walls 328 of the second chamber 324B have aconcave outer surface 328A.

Turning to FIG. 4D, the bioreactor 330 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 330 into afirst chamber 334A shaped as a tapering part of the bioreactor 330 and asecond chamber 334B shaped as a tapering part of the bioreactor 330. Thebioreactor 330 has a tapering shape. The tapering walls 338 of thebioreactor 330 have a convex outer surface 338A.

Turning to FIG. 4E, the bioreactor 340 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 340 into afirst chamber 344A shaped as a tapering part of the bioreactor 340 and asecond chamber 344B shaped as a tapering part of the bioreactor 300. Thebioreactor 340 has a tapering shape. The tapering walls 348 of thebioreactor 340 have a convex outer surface 348A.

Turning to FIG. 4F, the bioreactor 350 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 350 into afirst chamber 354A shaped as a conical of part of the bioreactor 350 anda second chamber 354B shaped as a frustoconical part of the bioreactor300. The bioreactor 350 has a conical shape.

Turning to FIG. 4G, the bioreactor 360 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 360 into afirst chamber 364A shaped as a cylindrical part of the bioreactor 360and a second chamber 364B shaped as a cylindrical part of the bioreactor360. The bioreactor 360 has a cylindrical shape.

Turning to FIG. 4H, the bioreactor 370 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 370 into afirst chamber 374A shaped as a hemispherical part of the bioreactor 370and a second chamber 374B shaped as a frustoconical part of thebioreactor 370. The bioreactor 370 has a shape similar to a chalice.

Turning to FIG. 4I, the bioreactor 380 includes the perforated barrier12 as disclosed hereinabove which divides the bioreactor 380 into afirst chamber 384A and a second chamber 384B. The bioreactor 380includes a vertical wall portion 380H that is orthogonal to the bottompart 380B of the bioreactor 380 (the wall portion 380H forms an angle of90 degrees with the bottom part 380B) and a slanted wall portion 380Ethat is slanted at an angle α1 relative to the wall portion 380H (thedashed line 385 is parallel to the vertical wall portion 38011).Typically the angle α1<90° and in some embodiments but not obligatorilyα1<45°.

Reference is now made to FIG. 4J, which is a top view of the bioreactor380 of FIG. 4I. The top part 380C of the bioreactor 380 is shaped suchthat it has a semi-circular portion 380E, two straight portions 380F and380G and a straight portion 380H. The bottom part 380B of the bioreactor380 (schematically illustrated by the dashed line 380B in FIG. 4J) canhave a shape or contour similar to the shape or contour of the top partbut has a smaller cross-sectional area than the cross-sectional area ofthe top part 380C due to the slanting of the wall portion 380E.

It is noted that while the shape of the top part 380C of the bioreactor380 is as disclosed hereinabove with respect to FIG. 4J, this is notobligatory and other different shapes of the top part 380C and thebottom part 380B can be used in some embodiment of the bioreactorshaving a slanted wall portion or part. In some embodiments of thebioreactors having a slanted wall portion and a non-slanted wallportion, the top and/or bottom parts of the bioreactor can have anyother desired shape including but not limited to, a semi-ellipticalshape, a semi-circular shape, a rectangular shape, a square shape, atrapezoidal shape, a polygonal shape, or any other suitable regular orirregular shape.

It is noted that while in several of the embodiments of the bioreactorsdisclosed hereinabove transversal cross sections of the bioreactor canbe circular, in other embodiment of the bioreactors of the presentapplication, transversal cross sections of the bioreactor can have othershapes, including, but not limited to an elliptical shape, a polygonalshape, a regular polygonal shape, or any other suitable shape.

It is further noted that in some of the bioreactors disclosed hereindifferent transversal cross sections taken at different positions alonga longitudinal axis of the bioreactor can have different shapes. Forexample, returning to FIG. 4C, while the transversal cross section takenalong the lines I-I and II-II (which are both orthogonal to thelongitudinal axis 335) can both be circular in shape, in accordance withanother embodiment of the bioreactor, the transversal cross sectiontaken along the line I-I can be circular in shape, and the transversalcross section taken along the line II-II can be elliptical in shape.

Furthermore, in accordance with some embodiments of the bioreactor, theshape of the bioreactor can be a conical shape, a frustoconical shape, atapering shape, a cylindrical shape, a polygonal prism shape, a taperingshape having an ellipsoidal transversal cross section, a tapering shapehaving a polygonal transversal cross section, a shape having acylindrical part and a tapering part, and a shape having a conical ortapered part and a hemispherical part.

Reference is now made to FIG. 5 which is a schematic block diagramillustrating the components of a bioreactor system, in accordance withsome embodiments of the bioreactor systems of the present application.The bioreactor system 400 includes a bioreactor 410, a pump 404, thebioreactor system can also include N+1 controllable valves 424A-424N(wherein N is an integer number) and another controllable valves 439.The bioreactor system can also include an (optional) controller 430, an(optional) fluid reservoir 420, an (optional) fluid impeller 418 an(optional) oxygenating system 460 and an (optional) heater/cooler unit470. In some embodiments, a bioreactor system disclosed herein furthercomprises a controller. In some embodiments, a bioreactor system furthercomprises a fluid reserve. In some embodiments, a bioreactor systemfurther comprises a fluid impeller. In some embodiments, a bioreactorsystem further comprises an oxygenating system. In some embodiments, abioreactor system further comprises a heater unit. In some embodiments,heating on the liquid medium can be provided via a heating jacket or anyprovided bioreactor surrounding environment (not shown). In someembodiments, a bioreactor system further comprises a cooler unit. Insome embodiments, a bioreactor system further comprises a heater unitand a cooler unit. According to some embodiments the liquid'stemperature can be controlled (heated/cooled to a desired temperature)at the liquid's reservoir.

In some embodiments, a bioreactor system comprises a control signal toan outlet valve (426). In some embodiments, a bioreactor systemcomprises a control signal (439A) for a pump.

According to some embodiments, the bioreactor 410 can be any of thebioreactors that have multiple fluid outlet ports (as disclosed in thepresent application and illustrated in the drawing figures) whichinclude a first (lower) chamber and a second (upper) chamber (the firstand second chambers are not shown in detail in the schematic blockdiagram of FIG. 5, but can be seen as illustrated, for example, in FIG.2). Each of the multiple fluid outlet ports opening into the secondchamber (not shown in the schematic diagram of FIG. 5 for the sake ofclarity) is fluidically connectable to a fluid manifold 428 through oneof the respective N valves 424A-424N.

According to some embodiments, the fluid manifold 428 is configured tofeed the growth medium collected from the second chamber of thebioreactor 410 to the pump 404 which is configured to pump the growthmedium back into the first chamber of the bioreactor 410 through thefluid inlet port 448 which opens into the first chamber of thebioreactor 410. The fluid input port 448 is configured to (optionally)feed the growth medium to the (optional) fluid impeller 418 as disclosedin detail hereinabove with respect to FIG. 2. The sensor unit 422 can beimplemented as disclosed hereinabove with respect to any of the sensorunits 22, 122 and 222 (of FIGS. 1, 2 and 3, respectively).

According to some embodiments, the fluid reservoir 420 can be a fluidreservoir external to the bioreactor 410, as disclosed hereinabove, andis configured to be fluidically and controllably coupled to the pump 404through the valve 439. Each of the N valves 404A-404N is suitablyconnected to the controller 430 by a respective communication lines429A-429N to receive control signals from the controller for opening orclosing any of the valves 424A-424N. The valve 439 is connected to thecontroller 430 by a suitable communication line for receiving controlsignals there from to open or close the valve 439 for allowing growthmedium to flow from the reservoir 420 into the pump 404 and there frominto the bioreactor 410 as disclosed in detail hereinabove for the valve39 (of FIG. 1).

According to some embodiments, the pump 404 is configured to be suitablyconnected to the controller 430 by a suitable communication line forcontrolling the operation of the pump 404. For example, such controlsignals can turn the pump on or off and can also control the rate offlow of growth medium through the pump 404 (or the rate of pumping ofthe growth medium by the pump 404.

According to some embodiments, the (optional) heater/cooler 470 isconfigured to be disposed in the bioreactor 410 (in some embodimentswithin the first chamber thereof) to heat or cool the growth mediumwithin the bioreactor 410 to maintain a desired temperature of thegrowth medium. Optionally, a water jacket (not shown) or blanket (notshown) or any other controlled temperature environment can be used fortemperature control of the bioreactor.

According to some embodiments, if the sensor unit 422 includes atemperature sensor, signals representing the sensed temperature can besent from the temperature sensor to the controller 430 through acommunication line(s) 422A. The controller 430 is configured to processsuch signals and send appropriate signals to the heater/cooler 470 formaintaining a desired temperature, or a set temperature or a presettemperature within the bioreactor as is well known in the art oftemperature control. Any other sensors included within the sensor unit422 are configured to (optionally) send through the communicationline(s) 422A sensor signals representing any sensed physical or chemicalparameter of the growth medium in the bioreactor 410, as disclosed indetail hereinabove.

According to some embodiments, the controller 430 is configured toprocess any such sensor signals to determine the status of the growthmedium and can also use the processed either display status data orabout any monitored or sensed physical or chemical parameters to anoperator or user of the bioreactor system 400 by an (optional) displayunit (not shown in detail in FIG. 5) included in an (optional) userinterface 431 included in the controller 430, as is disclosedhereinabove in detail.

For example, in a case in which the sensor unit includes a dissolvedoxygen sensor for sensing the amount of oxygen dissolved in the growthmedium within the bioreactor 430, the sensor signals can be processed bythe controller 430 and if the concentration of dissolved oxygen isdifferent than a desired set, preset, or predetermined) value, thecontroller 430 is configured to send control signals to the oxygenatingsystem 460 for stopping or starting the introducing of oxygen containinggas into the growth medium within the bioreactor 430 (or within thefluid reservoir 420, depending on the specific implementation of thebioreactor system 400 to suitably adjust the dissolved oxygen level tothe desired level.

It is noted that as disclosed in detail hereinabove with respect to thecontroller 30 (of FIG. 1), the controller unit 430 is configured toinclude any type of suitable processor (digital and/or analog) which canbe operated by suitable software to automatically or semi-automaticallycontrol the operation of the bioreactor 430 or at least some of theoperational functions thereof. For example, while the determining of thegrowth medium level and rate of flow within the second chamber of thebioreactor 410 can be set manually by an operator by using the userinterface 431, the regulation of the bioreactor's temperature and/ordissolved oxygen concentration within the growth medium can beautomatically controlled by suitable software operating on thecontroller 430. Similarly, the addition of amounts of fresh growthmedium from the reservoir 420 can be fully automated by periodicallydraining an amount of the growth medium from the first chamber through adraining port 427 by turning the 404 off and opening a draining valve425, and then closing the draining valve 425, opening the valve 439 andturning the pump 404 on to allow an amount of fresh growth medium to bepumped into the first chamber and then closing the valve 439 to restartthe recirculation of the growth medium through the bioreactor 410. Asimilar method can be used in the reservoir 430 resulting in mediarefreshment.

When the cells or microorganisms grown within the bioreactor need to beharvested, the harvesting can be performed is several different ways inaccordance with the specific structure of the bioreactor.

In some embodiments of the bioreactor (such as, for example in thebioreactor 10 of FIG. 1), the perforated barrier is fixed and immovablyattached to the walls of the bioreactor and the harvesting. Theharvesting of cells in such a bioreactor, can be performed by using oneor more harvesting ports disposed in the vessel walls of the bioreactorand opening into the second chamber in the vicinity of the upper surfaceof the perforated barrier (such as, for example, the single harvestingport 21 of the bioreactor 10 which opens into the second chamber 14B inthe vicinity of the surface 12A of the perforated barrier 12 of FIG. 1.However, since the flat surface 12A of the bioreactor 10 is horizontalduring harvesting, the harvesting may be somewhat hampered as some ofthe cells 3 may not reach the opening of the harvesting port 21.

Reference is now made to FIGS. 6A-6B which are schematic partcross-sectional diagrams illustrating two possible positional states ofa tiltable bioreactor, in accordance with some embodiments of thebioreactors of the present application.

It is noted that the bioreactor 510 of FIGS. 6A-6B is only schematicallyillustrated in outline and only the components necessary forunderstanding the harvesting operation thereof are shown in detail.Other components of the bioreactor 510 not necessary for understandingof the tilting action and the cell harvesting are not shown in FIGS.6A-6B for the sake of clarity of illustration and can be implemented asdisclosed in detail for the bioreactors of FIGS. 1-5 or any otherbioreactors disclosed herein. In the tiltable bioreactor 510 of FIG. 6A,the bioreactor includes vessel walls 510A, top part 510C and bottom part510B. The space within the bioreactor 510 is divided into a firstchamber 514A and a second chamber 514B by a perforated barrier 512. Anyother components of the bioreactor 510 not shown in detail in FIGS. 6A-Dcan be as disclosed in detail hereinabove with respect to the bioreactor10 of FIG. 1. In FIG. 6A, the bioreactor 510 is in a vertical state inwhich the longitudinal axis 535 of the bioreactor 510 is vertical (inFIG. 6A this is represented by the longitudinal axis 535 being alignedalong the vertical axis V). The bioreactor 510 includes a harvestingport 521 and a valve 523.

In FIG. 6A, the valve 523 is shown in the closed state and thebioreactor 510 is shown to contain a small amount of growth medium 2 inwhich the cells 3 to be harvested are suspended after most (but not all)of the growth medium 2 has been drained from the bioreactor 510 throughan outlet port 527 opening into the first chamber 514A by opening thevalve 525. During draining, according to some embodiments, some of thegrowth medium 2 held in the second chamber 514B passes downstreamthrough the perforations of the perforated barrier and into the firstchamber 514A and exits from the outlet port 527 but the cells 3 areretained in the second chamber 514B as they cannot pass through theperforations of the perforated barrier. According to some embodiments,the draining can also be provided via a deep tube (not shown) that canbe inserted to the upper chamber via for example one of the outlet ports126A-126D (shown in FIG. 2), as long as the deep tube is positionedabove cell mass concentration. According to some embodiments, thedraining can also be provided by opening the valve of one of the outletports 126A-126D (shown in FIG. 2), as long as the outlet port is locatedabove cell mass concentration. This results in concentrating the cellsin the second chamber 514B due to the reduction of the amount of growthmedium 2 remaining in the second chamber. When the level of the growthmedium 2 in the second chamber 514B has been sufficiently reduced, thevalve 525 can be closed.

According to some embodiments, in order to perform the cell harvesting,the bioreactor 510 is now tilted as illustrated in FIG. 6B, whichillustrates the bioreactor 510 in a tilted state. In the tilted state,the longitudinal axis 535 of the bioreactor 510 is tilted at an angle αto the vertical direction (represented in FIG. 6B by the vertical dashedline V). The angle α can be any convenient angle in the range 0<α<90degrees. After the bioreactor 510 is tilted (for example at an angleα=45 degrees), the suspended cells 3 can be harvested into a suitablecollecting vessel such as a test tube 511 by opening the valve 523 asillustrated in FIG. 6B. The advantage of such tiltable bioreactors isthat during harvesting, the yield of collected cells can be higher ascompared to the yield of harvesting performed in non-tiltablebioreactors such as the bioreactor 10 of FIG. 1. As FIGS. 6B, 6C, and 6Dare embodiments of the bioreactor 510 of FIG. 6A, the elements in FIGS.6B, 6C, and 6D that are identified above for FIG. 6A have the samemeaning and qualities as these elements in FIG. 6A.

According to some embodiments, the tilting action of the bioreactor 510(or of any other type of tiltable bioreactor implemented as disclosed inthe present application) can be performed by any mechanical means knownin the art, such as, but not limited to, by tilting the bioreactorwithin any mechanical support structure (not shown) holding thebioreactor 510. Additionally, in accordance with some additionalembodiments of the bioreactor, the bioreactor 510 is configured to betiltably supported within a fork-like gantry (not shown) having twoopposing arms tiltably holding a bracket within which the bioreactor 510can be supported. Such mechanical structures for tiltably holding avessel such that it can be vertically aligned or tilted at any desiredangle to the vertical are well known in the art, and are therefore notdescribed in detail hereinafter.

Reference is now made to FIGS. 6C and 6D which are schematic partcross-sectional views illustrating a bioreactor having a fixed slantedperforated barrier, in accordance with some embodiments of thebioreactors of the present application;

It is noted that the bioreactor 550 of FIGS. 6C-6D is only schematicallyillustrated in outline and only the components necessary forunderstanding the harvesting operation thereof are shown in detail.Other components of the bioreactor 550 that are not necessary forunderstanding of the cell harvesting method are not shown in FIGS. 6C-6Dfor the sake of clarity of illustration and can be implemented asdisclosed in detail for the bioreactors of FIGS. 1-2 and 5 or any otherbioreactors disclosed herein.

The bioreactor 550 of FIG. 6C includes vessel walls 550A, a top part550C and a bottom part 550B. The space within the bioreactor 550 isdivided into a first chamber 520A and a second chamber 520B by aperforated barrier 512. Any other components of the bioreactor 550 notshown in detail in FIGS. 6C and 6D are disclosed in detail hereinabovewith respect to the bioreactor 10 of FIG. 1. The perforated barrier 522is sealingly and fixedly attached to the vessel walls 550A and isslanted at an angle β relative to the horizontal plane H of thebioreactor 550 (the horizontal plane is schematically represented by thedashed line H in FIGS. 6C and 6D). The angle β can be any angle in therange 0.2<β<45 degrees, but other angles smaller or larger than thisrange can be used, depending, inter alia, upon the application. Intypical applications the angle β can be in the range of 0.2<β<15degrees.

The bioreactor 550 includes a harvesting port 531 having a valve 533.The valve 533 of the harvesting port 531 is illustrated in FIG. 6C in aclosed state and the bioreactor 550 is shown to contain an amount ofgrowth medium 2 including the cells 3 suspended in the growth medium 2.

Turning now to FIG. 6D, when the cells 3 need to be harvested, most (butnot all) of the growth medium 2 is drained from the bioreactor 550through an outlet port 527 opening into the first chamber 520A byopening the valve 525 of the outlet port 527. During draining, most ofthe growth medium 2 (or a washing buffer used to wash the cells 3) flowsinto the first chamber 520A by passing through the perforations in theperforated barrier 522 and exits from the outlet port 527 but the cells3 are retained in the second chamber 520B as they cannot pass throughthe perforations in the perforated barrier. This results inconcentrating the cells 3 in the second chamber 520B due to thereduction of the amount of growth medium 2 remaining in the secondchamber 520B. When the level of the growth medium 2 in the secondchamber 520B has been sufficiently reduced, the valve 525 can be closed.

Turning to FIG. 6D, the bioreactor 550 is illustrated with the secondchamber 520B containing the cells 3 concentrated in the small amount ofthe growth medium 2 remaining within the second chamber 520B after mostof the growth medium 2 was drained from the second chamber 520B; forexample, by opening the valve 525 of the outlet port until the desiredamount of growth medium is drained from the bioreactor 550 and thenclosing the valve 525, and/or via the deep tube (as mentioned above)and/or one of the second chamber's outlet ports (as mentioned above).The harvesting of the cells can be performed by opening the valve 533 ofthe harvesting port 531 and connecting a collecting vessel 511 to theend of the harvesting port 531.

Reference is now made to FIGS. 7-9, which are schematic, partcross-sectional diagrams illustrating three different embodiments ofbioreactors including three different types of non-planar (not flat)perforated barriers, in accordance with some embodiments of thebioreactors of the present application. It is noted that for the sake ofclarity of illustration, the schematic drawings of FIGS. 7-9 illustrateonly the general shape of the walls of the bioreactors and the shape ofthe perforated barrier included therein and of the harvesting portassociated with the perforated barrier and do not show any details ofany additional components of the bioreactors or bioreactor systems (suchas, for example, various openings in the walls of the bioreactors,sensor units, fluid inlet ports, fluid outlet ports, draining ports,harvesting ports, heating units, cooling units, fluid impellers, gasdispersing heads, valves, pumps, controllers, self-sealable gaskets,fluid manifolds or any other components) which are not important tounderstanding the shape of the perforated barriers shown of thebioreactors. It will be appreciated by those skilled in the art that anysuch components which are not shown in FIGS. 7-9, can be included in anynon-mutually exclusive combinations and/or permutations in any of thebioreactors schematically illustrated in FIGS. 7-9, as is disclosedherein in detail herein and as illustrated in the drawing figures.

Turning to FIG. 7, the bioreactor 610 has vessel walls 610A, a curvedperforated bather 612 is fixedly (non-movably) and sealingly attached tothe vessel walls 610A, dividing the space within the bioreactor 610 intoa first chamber 614A and a second chamber 614B. The bioreactor 610further comprises a harvesting port 627 which is a hollow member thatincludes a valve 623. The harvesting port 627 is similar in structure tothe harvesting port 127 of FIG. 2. The harvesting port 627 is sealinglyattached to the curved perforated barrier 612 and opens at the surface612A into the second chamber 614B.

As disclosed in detail hereinabove for the harvesting port 127 (of FIG.2), the harvesting port 627 sealingly passes through the vessel walls610A to exit the bioreactor 610. The upper surface 612A of the curvedperforated bather 612 facing the top part 610C of the bioreactor 610 isconcave, which can advantageously increase the yield of harvested cellsas compared to the yield of harvested cells in a bioreactor having afixed (non-movable) flat (planar) perforated barrier (such as, forexample, the bioreactor 110 of FIG. 2).

Turning to FIG. 8, the bioreactor 710 has vessel walls 710A, a conicalperforated bather 712 is fixedly (non-movably) and sealingly attached tothe vessel walls 710A, dividing the space within the bioreactor 710 intoa first chamber 714A and a second chamber 714B. The bioreactor 710further comprises a harvesting port 727 which is a hollow member thatincludes a valve 723. The harvesting port 727 is similar in structure tothe harvesting port 127 of FIG. 2. H represents the horizontal plane Hof the bioreactor (710).

According to some embodiments, the harvesting port 727 is sealinglyattached to the conical perforated barrier 712 and opens at the surface712A into the second chamber 714B. As disclosed in detail hereinabovefor the harvesting port 127 (of FIG. 2), the harvesting port 727sealingly passes through the vessel walls 710A to exit the bioreactor710. The upper surface 712A of the conical perforated barrier 712 facingthe top part 710C of the bioreactor 710 is a conical surface, which canadvantageously increase the yield of harvested cells as compared to theyield of harvested cells in a bioreactor having a fixed (non-movable)flat (planar) perforated barrier (such as, for example, the bioreactor110 of FIG. 2).

Turning to FIG. 9, the bioreactor 810 has vessel walls 810A, a taperingperforated barrier 812 is fixedly (non-movably) and sealingly attachedto the vessel walls 810A, dividing the space within the bioreactor 810into a first chamber 814A and a second chamber 814B. The bioreactor 810further comprises a harvesting port 827 which is a hollow member thatincludes a valve 823. The harvesting port 827 is similar in structure tothe harvesting port 127 of FIG. 2. The harvesting port 827 is sealinglyattached to the tapering perforated barrier 812 and opens at the surface812A into the second chamber 814B.

As disclosed in detail hereinabove for the harvesting port 127 (of FIG.2), the harvesting port 827 sealingly passes through the vessel walls810A to exit the bioreactor 810. The upper surface 812A of the taperingperforated barrier 812 facing the top part 810C of the bioreactor 810 isa tapering surface, which can advantageously increase the yield ofharvested cells as compared to the yield of harvested cells in abioreactor having a fixed (non-movable) flat (planar) perforated barrier(such as, for example, the bioreactor 110 of FIG. 2).

It is noted that while all the bioreactors disclosed hereinabove andillustrated in FIGS. 1-3, 4A-4I, 6A-6B and 7-9 include fixed non-movableperforated barriers, this is not obligatory to practicing the using thebioreactors or systems thereof disclosed herein, and in accordance withsome embodiments, the bioreactors are configured to include movable(non-fixed) perforated barriers or tiltable perforated barriers.

Reference is now made to FIGS. 10A-10B, 11A-11B and 12A-12B, whichillustrated some embodiments of reactors having movable and/or tiltableperforated barriers. FIGS. 10A-10B are schematic part cross-sectionaldiagrams illustrating two different states of a bioreactor including adeformable perforated barrier, in accordance with some embodiments ofthe bioreactors of the present application.

FIGS. 11A-11B are schematic part cross-sectional diagrams illustratingtwo different states of a bioreactor including a buckling perforatedbarrier, in accordance with some embodiments of the bioreactors of thepresent application, and FIGS. 12A-12B are schematic partcross-sectional diagrams illustrating two different states of abioreactor including a tiltable perforated barrier, in accordance withsome embodiments of the bioreactors of the present application. It isnoted that, for the sake of clarity of illustration, the schematicdrawings of FIGS. 10A-10B, 11A-11B and 12A-12B, illustrate only thegeneral shape of the walls of the bioreactors and the shape andarrangement of the movable or deformable or tiltable or bucklingperforated barrier included therein and of the harvesting portassociated with the perforated barrier and do not show any details ofany additional components of the bioreactors or bioreactor systems (suchas, for example, various openings in the walls of the bioreactors,sensor units, fluid inlet ports, fluid outlet ports, draining ports,harvesting ports, heating units, cooling units, fluid impellers, gasdispersing heads, valves, pumps, controllers, self-sealable gaskets,fluid manifolds or any other components) which are not important tounderstanding the shape of the perforated barriers shown of thebioreactors. It will be appreciated by those skilled in the art that anysuch components which not shown in FIGS. 10A-10B, 11A-11B, and 12A-12Bcan be included in any non mutually exclusive combinations and/orpermutations in any of the bioreactors schematically illustrated inFIGS. 10A-10B, 11A-11B, 12A-12B, and 13 as is disclosed in detail hereinand as illustrated in the drawing figures.

Turning now to FIGS. 10A-10B, the bioreactor 910 has vessel walls 910A,a deformable perforated barrier 912 is fixedly and sealingly attached tothe vessel walls 910A, dividing the space within the bioreactor 910 intoa first chamber 914A and a second chamber 914B. The deformableperforated barrier 912 includes multiple perforations as disclosed indetail hereinabove and allows the growth medium 2 to bidirectionallypass there through (from the first chamber 914A to the second chamber914B, and vice versa) but blocks the passage of cells or organisms therethrough as is disclosed in detail hereinabove. According to someembodiments, the perforated barrier 912 can be made from a material thatis biocompatible for the growing of cells or microorganisms and is alsoflexible or deformable such that a force applied to the perforatedbarrier 912 can deform its shape.

The bioreactor 910 further comprises a harvesting port 927 which is ahollow member that includes a valve 923. The harvesting port 927 issealingly attached to the deformable perforated barrier 912 and opens atthe surface 912A into the second chamber 914B. The harvesting port 927sealingly passes through the vessel walls 910A to exit the bioreactor910. The harvesting port 927 is a hollow member that has a first rigid(non movable) part (or portion) 927A disposed within the first chamber914A. The first rigid part 927A sealingly passes through the vesselwalls 910A and exits outside the bioreactor 910. The first rigid part927A has a valve 923 therein for opening or closing the harvesting port927. According to some embodiments, the harvesting port 927 furthercomprises a second flexible and/or compressible part (or portion) 927Bwhich is sealingly attached to the first part 927A at one end thereof.The flexible and/or compressible part 927B and the rigid part 927A areconnected together to form the hollow member opening to the secondchamber 914B at the end of the flexible part 927B which is sealinglyattached to the deformable perforated barrier 912 and open at thesurface 912A thereof.

It is noted that while the harvesting ports disclosed in someembodiments of the present application are open at the upper surface ofthe perforated barrier, alternative embodiments can include harvestingports which are closed or sealed at their end connected to theperforated barrier by a thin sealing membrane (not shown). In suchembodiments, when the harvesting port needs to be used for harvestingcells from the second chamber of the bioreactor, the sealing membrane isconfigured to burst open by either inserting a sharp sterile wire-likeinstrument through the harvesting port and bursting the sealingmembrane, or by inserting a sharp sterile instrument through any of theopenings in the top part of the bioreactor into the second chamber andbursting the sealing membrane. Any other mechanical or magneticmechanisms can also be used for bursting the sealing membrane of suchsealed harvesting ports as is known in the art.

According to some embodiments, the bioreactor 910 includes a magneticmember 915 attached to the second compressible (or flexible part) 927B,as illustrated in FIGS. 10A-10B. Alternatively, in accordance with yetanother embodiment of the bioreactor 910, the magnetic member 915 isconfigured to be attached to the deformable perforated barrier 912, insome embodiments near the central part of the perforated barrier 912(not shown in FIGS. 10A-10B). The magnetic member 915 is configured tobe (optionally) shaped like an annular member made from a permanentlymagnetized material.

For example, the magnetic member 915 can be made from a FeNdB (lionNeodymium Boron) permanent magnet, a samarium-cobalt permanent magnet orany other magnetic or paramagnetic material known in the art such as,for example, Iron. If necessary, the magnetic member 915 can be coatedwith, or embedded in a biocompatible material such as, for example, abiocompatible plastic or any suitable biocompatible polymer basedmaterial, a biocompatible ceramic layer or any other suitablebiocompatible and (in some embodiments) sterilizable material.

Turning now to FIG. 10B, when the cells 3 need to be harvested from thebioreactor 910, an amount of growth medium 2 can be drained from thefirst chamber 914A of the bioreactor 910 through a suitable outlet port(not shown in FIGS. 10A-10B, for the sake of clarity of illustration,but similar to the outlet port 27 of FIG. 1 or to the outlet port 227 ofFIG. 3) as disclosed hereinabove for concentrating the cells 3 in theremaining growth medium 2. A strong magnet M can then be suitably placednear the bioreactor 910 as illustrated in FIG. 10B. The magnet M can beany suitable permanent magnet or an electromagnet known in the art. Theplacement of the magnet M near the bioreactor 910 exerts a magneticforce represented by the arrows F which is directed towards the magnetM. The force pulls the second part 927B downwards causing the deformableperforated barrier 912 attached to the second compressible part 927 tobe also pulled downwards and to deform.

When the magnetic force is acting on the second compressible (orflexible or shortenable) part 927B, the second compressible part 927 iscompressed such that it's length shortens, allowing the part of theperforated barrier 912 attached to the second part 927B to movedownwards, causing the shape of the perforated barrier to deform into adeformed state (as illustrated in FIG. 10B). The deformation of thedeformable perforated barrier 912, results in the perforated barrier 912assuming a slightly curved shape, such that the upper surface 912A ofthe perforated barrier 912 in the deformed state can nearly resemble aparabolloidal surface.

Returning to FIG. 10A, the bioreactor 910 is shown with the deformableperforated barrier 912 in a flat non-deformed state. In thisnon-deformed state, the upper surface 912A of the perforated barrier 912is substantially planar (flat). In this state the cells 3 can be grownin the second chamber 914B as is described in detail for otherbioreactor embodiments disclosed hereinabove.

Returning now to FIG. 10B, the bioreactor 910 is illustrated with thedeformable perforated barrier 912 in a deformed state. In this deformedstate, the upper surface 912A of the perforated barrier 912 is a curvedsurface. In this deformed state, the concentrated cells 3 suspended inthe growth medium 2 can be harvested by opening the valve 923 of theharvesting port 927 and collecting the cell 3 suspended in the growthmedium 2 into a collection vessel 511 as disclosed hereinabove. Theconcave surface 912A of the curved shape of the deformed perforatedbarrier 912 can advantageously increase the yield of harvested cells ascompared to the yield of harvested cells in a bioreactor having a fixed(non-movable) flat (planar) perforated barrier (such as, for example,the bioreactor 110 of FIG. 2).

Turning now to FIGS. 11A-11B, the bioreactor 1010 has vessel walls1010A. A buckling perforated barrier 1012 is fixedly and sealinglyattached to the vessel walls 1010A, dividing the space within thebioreactor 1010 into a first chamber 1014A and a second chamber 1014B.The buckling perforated barrier 1012 includes multiple perforations asdisclosed in detail hereinabove and allows the growth medium 2 tobidirectionally pass there through (from the first chamber 1014A to thesecond chamber 1014B, and vice versa) but blocks the passage of cells ormicroorganisms there through as is disclosed in detail hereinabove.According to some embodiments, the buckling perforated barrier 1012 canbe made from a stiff but flexible material which is biocompatible forthe growing of cells or microorganisms.

According to some embodiments, the perimeter of the buckling perforatedbarrier 1012 is sealingly attached to the vessel walls 1010A such thatin a first stable state of the buckling perforated barrier (illustratedin FIG. 11A), the perforated barrier 1012 is convex in shape and theupper surface 1012A of the perforated barrier 1012 which faces the toppart 1010C of the bioreactor 1010 is a convex surface. According to someembodiments, if a force of sufficient magnitude is applied to thebuckling perforated barrier 1012, the buckling perforated barrier 1012will flip into a second stable state (illustrated in FIG. 11B). Ascompared with the barrier (1012) in FIG. 11A, the barrier (1012) in FIG.11B is tilted a bit towards the bottom of the bioreactor vessel. In thesecond state of the perforated barrier 1012, the perforated barrier 1012is concave in shape and the upper surface 1012A of the perforatedbarrier 1012 which faces the top part 1010C of the bioreactor 1010 is aconcave surface.

According to some embodiments, the buckling perforated barrier 1012 isconfigured such that it is in a bi-stable configuration in which atransition between the two stable states of the buckling perforatedbarrier requires the application of sufficient force to the perforatedbarrier 1012. According to some embodiments, the bioreactor 910 furthercomprises the harvesting port 927 which is a hollow member that includesa valve 923. The harvesting port 927 is sealingly attached to thebuckling perforated barrier 1012 and opens at the upper surface 1012Ainto the second chamber 1014B. The harvesting port 927 sealingly passesthrough the vessel walls 1010A to exit the bioreactor 1010. Theharvesting port 927 is a hollow member that has a first rigid(non-movable) part (or portion) 927A disposed within the first chamber1014A.

According to some embodiments, the first rigid part 927A sealinglypasses through the vessel walls 1010A and exits outside the bioreactor1010. The first rigid part 927A has a valve 923 therein for opening orclosing the harvesting port 927. According to some embodiments, theharvesting port 927 further comprises a second flexible and/orcompressible part (or portion) 927B which is sealingly attached to thefirst part 927A at an end thereof. According to some embodiments, theflexible and/or compressible part 927B and the rigid part 927A areconnected together to form the hollow member opening to the secondchamber 1014B at the end of the flexible part 927B which is sealinglyattached to the buckling perforated barrier 1012 and open at the surface1012A thereof.

According to some embodiments, the bioreactor 1010 includes a magneticmember 1015. The magnetic member 1015 is configured to (optionally) havean annular shaped magnetic member attached to the deformable perforatedbarrier 1012, as illustrated in FIGS. 11A-11B. Alternatively, inaccordance with yet another embodiment of the bioreactor 1010, themagnetic member 1015 is configured to be attached to the secondcompressible (or flexible) part 927B of the harvesting port 927 (thisembodiment is not shown in FIGS. 10A-10B). However, the magnetic member1015 can have any other shape suitable for applying an appropriatelydownward directed force to the buckling perforated barrier or to thesecond compressible (or flexible) part 927B of the harvesting port 927(depending on the part to which the magnetic member 1015 is attached inthe above disclosed different alternative embodiments).

According to some embodiments, the magnetic member 1015 can be made froma permanently magnetized material or from a paramagnetic material orfrom any other magnetizable material as disclosed hereinabove in detailwith respect to the magnetic member 1015. If necessary, the magneticmember 1015 can be coated with or embedded in a biocompatible materialsuch as a biocompatible plastic or any suitable biocompatible polymerbased material, a biocompatible ceramic layer or any other suitablebiocompatible and (in some embodiments) sterilizable material, asdisclosed hereinabove with respect to the magnetic member 915.

Turning now to FIG. 11B, when cells (not shown) need to be harvestedfrom the bioreactor 1010, an amount of growth medium (not shown) can bedrained from the first chamber 1014A of the bioreactor 1010 through asuitable outlet port (not shown in FIGS. 11A-11B, for the sake ofclarity of illustration, but similar to the outlet port 27 of FIG. 1 orto the outlet port 227 of FIG. 3) as disclosed hereinabove forconcentrating the cells in the remaining growth medium.

According to some embodiments, a magnet M is configured to then besuitably placed near the bioreactor 1010 as illustrated in FIG. 11B. Themagnet M can be any suitable permanent magnet or an electromagnet knownin the art, as disclosed in detail with respect to FIG. 10B hereinabove.The placement of the magnet M near the bioreactor 1010 exerts a magneticforce on the magnetic member 1015 represented by the arrows F which isdirected towards the magnet M. The force pulls the buckling perforatedbarrier 1012 downward in the direction represented by the arrows F.According to some embodiments, the magnetic force is of a magnitude thatis more than sufficient to cause the buckling perforated barrier 1012 toflip from the first stable (convex) state to the second stable (concave)state (as is illustrated in FIGS. 11A-11B). According to someembodiments, when the perforated barrier 1012 flips from the first stateto the second state, the central part of the buckling perforated barrier1012 moves downwards and causes the second compressible part 927B to becompressed such that the length of the part 927B shortens, allowing thepart of the buckling perforated barrier 1012 attached to the second part927B to move downwards.

According to some embodiments, the flipping of the buckling perforatedbarrier 1012 from the first state to the second state can also beachieved mechanically using a weal (not shown) or a vertical rod-likepushing/pulling member (not shown) which is configured to be attached atone end thereof to the buckling perforated barrier 1012 while the secondend thereof sealingly and slidably passes through a suitable sealinggasket (not shown) disposed in an opening (not Shown) in the top part1010C of the bioreactor 1010.

According to some embodiments, when the buckling perforated barrier 1012is in the first state, pushing such a pushing/puling member downwards isconfigured to flip the buckling perforated barrier 1012 from the firststate to the second state. However, it will be appreciated by thoseskilled in the art that any other mechanical or magnetic orelectromagnetic mechanism or combinations of such mechanisms can be usedto flip the buckling perforated barrier from the first state into thesecond state and all such mechanisms or combinations of mechanisms aredeemed to be included within the scope of the embodiments of the presentapplication.

In FIG. 11B the bioreactor 1010 is illustrated with the bucklingperforated barrier 1012 in the second stable state. In this secondstate, the upper surface 1012A of the buckling perforated barrier 1012is a concavely curved surface. In this state, the concentrated cells(not shown) suspended in the growth medium (not shown) within the secondchamber 1014B can be harvested by opening the valve 923 of theharvesting port 927 and collecting the cell suspension into a collectionvessel (not shown) as disclosed hereinabove. According to someembodiments, the concave surface 1012A of the buckling perforatedbarrier 1012 in the second stable state can advantageously increase theyield of harvested cells as compared to the yield of harvested cells ina bioreactor having a fixed (non-movable) flat (planar) perforatedbarrier (such as, for example, the bioreactor 110 of FIG. 2).

Turning now to FIGS. 12A-12B, the bioreactor 1110 has vessel walls1110A. A tiltable perforated barrier 1112 is sealingly attached to thevessel walls 1110A, dividing the space within the bioreactor 1110 into afirst chamber 1114A and a second chamber 1114B. The perimeter of thetiltable perforated barrier 1112 is sealingly attached to a flexibleand/or deformable and/or stretchable annular member 1113. Typically, theannular sheet 1113 does not have any perforations therein. The annularmember 1113 can be made from a flexible or pliable and/or stretchablematerial, such as, for example, rubber or latex or a flexible polysilanebased thin material and is also sealably attached to the vessel walls1110A of the bioreactor 1110. In some embodiments, the annular membercan be non-permeable to either the cells 3 and to the growth medium 2.

The tiltable perforated barrier 1112 has multiple perforations thereinas disclosed in detail hereinabove and allows the growth medium 2 tobi-directionally pass there through (from the first chamber 1114A to thesecond chamber 1114B and vice versa) but blocks the passage of cells ormicroorganisms there through as is disclosed in detail hereinabove.According to some embodiments, the perforated barrier 1112 can be(optionally) made from a stiff or rigid material which is biocompatiblefor the growing of cells or microorganisms.

According to some embodiments, the bioreactor 1110 further comprises theharvesting port 1127 which is a hollow member that includes a valve1123. A first end 1127A of the harvesting port 1127 is disposed withinthe first chamber 1114A and is sealingly attached to the annular member1113 such that the end 1127A opens into the second chamber 1114B throughan opening 1113B on the upper surface 1113A of the annular member 1113.The harvesting port 1127 sealingly passes through the vessel walls 1110Ato exit the bioreactor 1110. The harvesting port 1127 is a hollowmember. A second end 1127B of the harvesting port 1127 is disposedoutside the bioreactor 1110 and includes a valve 1123 therein foropening or closing the harvesting port 1127.

According to some embodiments, the bioreactor 1110 also includes amagnetic member 1115. The magnetic member 1115 is configured to(optionally) be a bar shaped magnetic member attached to the perforatedbarrier 1112 near the perimeter of the perforated barrier 1112, asillustrated in FIGS. 12A-12B. However, the magnetic member 1115 can haveany other shape suitable for applying an appropriately downward directedforce to the tiltable perforated barrier 1112. When no force is appliedto the tiltable perforated barrier 1112, the perforated barrier 1112 ishorizontal or nearly horizontal as illustrated in FIG. 12A.

According to some embodiments, the magnetic member 1115 can be made froma permanently magnetized material or from a paramagnetic material or aferromagnetic material or from any other magnetizable material and can(optionally) be coated with or embedded in a biocompatible material, asdisclosed hereinabove in detail with respect to the magnetic member 915.

Turning to FIG. 12B, when the cells 3 need to be harvested from thebioreactor 1110, an amount of growth medium (not shown) can be drainedfrom the first chamber 1114A of the bioreactor 1110 through a suitableoutlet port (not shown in FIGS. 12A-12B, for the sake of clarity ofillustration, but similar to the outlet port 27 of FIG. 1 or to theoutlet port 227 of FIG. 3) as disclosed hereinabove for concentratingthe cells in the remaining growth medium 2. A magnet M can be suitablyplaced near the bioreactor 1110 as illustrated in FIG. 12B. The magnet Mcan be any suitable permanent magnet or an electromagnet known in theart, as disclosed in detail with respect to FIG. 10B hereinabove.

According to some embodiments, the placement of the magnet M near thebioreactor 1110 exerts a magnetic force on the magnetic member 1115represented by the arrow F which is directed towards the magnet M. Themagnetic force pulls the side 1112B of the perforated barrier 1112 towhich the magnetic member is attached downwards in the directionrepresented by the arrows F. As a result of the applied magnetic forceF, the perforated barrier 1112 is tilted such that the side 1112B of theperforated barrier 1112 is lower than the side 1112A of the perforatedmember 1112.

In FIG. 12B, the bioreactor 1110 is illustrated with the perforatedbarrier 1112 in a tilted state after a magnetic force has been appliedby the magnet M to the magnetic member 1115. In this tilted state, theconcentrated cells 3 suspended in the growth medium 2 within the secondchamber 1114B can be harvested by opening the valve 1123 of theharvesting port 1127 and collecting the cell suspension into acollection vessel (not shown) as disclosed hereinabove. The tilt(relative to the horizon) of the tiltable perforated barrier 1112 canadvantageously increase the yield of harvested cells as compared to theyield of harvested cells in a bioreactor having a fixed (non-movable)flat (planar) perforated barrier (such as, for example, the bioreactor110 of FIG. 2).

It is noted that during operating the bioreactors and bioreactor systemsof the present application, a liquid, e.g., a growth medium can besupplied by perfusion (constant replacement of media by recirculation asdisclosed in detail), or by fed batch (addition of specific nutrients tothe growth medium 2) or by batch (replacement of the growth medium orpart of the growth medium periodically if needed).

According to some embodiments, during harvesting of thecells/microorganisms grown in the bioreactors of the presentapplication, a need may arise to further concentrate the cells beingharvested. Such concentrating can be achieved without needing to performadditional actions outside the bioreactor (such as, for example,centrifugation in a centrifuge) which can adversely increase theprobability of contaminating the harvested cells by using an inlineconcentrating filter connected to the harvesting port.

According to some embodiments, washing of the cells in the bioreactorscan be done performed by replacing the growth medium 2 with a washbuffer as is known in the art. The replacement of the growth medium 2can be performed by draining the growth medium 2 from the bioreactor andfilling the bioreactor with new wash buffer several times. According tosome embodiments, the draining can be performed by using any of thedraining ports included in the first (lower) chamber of any of thebioreactors (such as, for example, the outlet port 27 of the bioreactor10 of FIG. 1, or the outlet port 227 of the bioreactor 210 of FIG. 3) orby using the output ports opening into the second (upper) chamberincluded in bioreactor embodiments that allow controlling of the levelof growth medium in the second chamber of the bioreactors (such as, forexample, the outlet port 126D of the bioreactor 110 of FIG. 2).

According to some embodiments, the bioreactors of the presentapplication are configured to allow cell separation and/or cellselection. Cell separation such as magnetic bead binding or antibodybinding can be performed inside the second chamber of some embodimentsof the bioreactors by using magnetic bead methods as is well known inthe art. According to some embodiments, magnetic beads (such as, forexample magnetic cell specific antibody-coated beads can be insertedinto the second chamber through any of the closable openings at the toppart of the bioreactors (such as, for example through the opening 110Eof the bioreactor 110 of FIG. 2). According to some embodiments, oncethe cells are bonded to the beads, the beads can be collected by using amagnet as is well known in the art, or by using a large filter that isadapted for selecting between the bead size and cells. Such filters canbe positive or negative selectors based on the filter's pore size. ForExample, cells attached to beads will not pass the filter whereas nativecells not attached to beads will pass through the pores in the filter.

Optionally, according to some embodiments the filter is configured tohave an affinity to the beads and can retain the beads and the cellsattached to the beads on the filter, while allowing unattached cells topass through the filter. Alternatively, it is possible to use a “teabag” shaped enclosure enclosing beads coated with a cell specificantibody that allows free passage of unbound cells through the pores inthe “tea bag” but retains any antibody coated beads and the cells thatare bonded to the beads within the “tea bag”. According to someembodiments, cells can pass through the “tea bag” membrane but the beadsare bigger and stay in the bag. According to some embodiments, cellsthat are attached to the beads can be retained in the “tea bag” andtaken out of the bioreactor or can be retained depending on the intendeduse and application.

According to some embodiments, the bioreactor can further comprise a 3Dhollow container (for example but not limited to a column-like container560) in its upper chamber (demonstrated in FIG. 6A), configured to beused for cell sorting; for a non-limiting example, precipitating CAR-Tcells with magnetic beads.

In some embodiments, the upper chamber (second chamber) is configured tocomprise an immobilized matrix and or beads in order to select cells ormicroorganisms having a particular binding activity. In some embodiment,the cells or microorganisms comprised in the fluid, for example but notlimited to a growth media or wash media, can be circulated through aninner 3D container comprising the immobilized matrix or beads. In someembodiments, the container walls permit cell and media flow in and outof the container but beads and cells bound to beads or the immobilizedmatrix are not permitted egress from the container. In some embodiments,the container comprises an immobilized matrix.

In some embodiments, beads comprise an affinity molecule on theirsurface. In some embodiments, an affinity molecule comprises apolypeptide, or portion thereof or a peptide or a carbohydrate bindingmolecule. In some embodiments, an affinity molecule comprises anantibody, biotin, avidin, a receptor or part thereof, an agglutinin, alectin, or any other molecule known in the art to which a cell ormicroorganism can bind. In some embodiments, the beads comprise magneticbeads. In the case of a magnet, magnetic beads can be retained in thecontainer by positioning a magnet near the container and retaining thepositive cells attached to the magnetic beads in the container whilecirculating back the negative cells.

In some embodiments, an immobilized matrix comprises an affinitymolecule on its surface. In some embodiments, an affinity moleculecomprises a polypeptide, or portion thereof or a peptide or acarbohydrate binding molecule. In some embodiments, an affinity moleculecomprises an antibody, biotin, avidin, a receptor or part thereof, anagglutinin, a lectin, or any other molecule known in the art to which acell or microorganism can bind.

In some embodiments, cells pass through the container, wherein if thecells or microorganism possess a binding partner to the surface markerpresent on the beads or immobilized matrix, the cells can bind to thesurface of the beads or immobilized matrix and be retained within thecontainer.

In some embodiments, the container comprises a “tea bag” like structure,wherein the sides are configured to be flexible.

According to some embodiments, a material such as Retro-Nectin can beadded to the barrier or to the affinity matrix in order to enhanceinfection rate of viruses, such as retor or lenti virus, as commonlyused for CAR T. According to some embodiments, the barrier and/or theaffinity matrix can be coated with relevant antibodies.

Activation of cells such as, for example, T cells can be achieved byadding cytokines and activation signals to the growth medium 2 or byco-culturing the T-cells with cytokine secreting cells that can beadhered to the perforated barrier or to any other type of suitablecarrier, or adhered to a “tea bag” or floating in a “tea bag” or onmagnetic beads, as disclosed hereinabove. Additionally, the activationof T-cells can be performed by co-culturing T-cells with Antigenpresenting cells, as is known in the art. It is noted that co-culturingof different types of cells is not limited to cell activation only. Fora non-limiting example, anti CD3/CD28 conjugated beads can also be usedto activate T cells. In another non-limiting example, Anti CD3 and AntiCD28 antibodies can also be used for activating T cells.

According to some embodiments, the bioreactors of the presentapplication are configured to also be used for co-culturing other typesof cells for achieving other results. For example, when culturingembryonic stem cells, the bioreactors of the present application areconfigured to also be used to co-culture the embryonic stem cells withfeeder cells (such as, for example, fibroblasts) which can release intothe growth medium substances and/or factors necessary for maintaininggrowth and proliferation of the stem cells and/or for inducingdifferentiation of the stem cells.

It is noted that for increasing harvesting efficiency the entire second(upper) chamber of the bioreactors disclosed hereinabove or the uppersurface of the perforated barriers included within such bioreactors canbe washed by growth medium can be perfused or added to the secondchamber of the bioreactors from the top or bottom of the second chamber(such as, for example by adding growth medium through the opening 110Eof the bioreactor 110, or through the opening 10G at the top part 10C ofthe bioreactor 10 of FIG. 1, or by injecting growth medium through theself-sealing gasket 211 of the bioreactor 210 of FIG. 3 by using asyringe filled with sterile growth medium 2). Such washing of the wallsof the second chamber and/or of the perforated barriers can result inpushing the cells towards the opening of any harvesting port openinginto the second (upper) chamber of the bioreactor as disclosedhereinabove.

According to some embodiments, cells that are grown within thebioreactors disclosed in the present application can be counted on lineand concentrated by using a circulation loop with a conic shapedconcentrating filter to allow volume reduction. The cell counting can beperformed by indirect measurements such as by using capacitancemeasurements, optical density measurements, and/or other optical sensorsas is well known in the art.

According to some embodiments, the bioreactors of the presentapplication are configured to allow culturing of adherent cells on anattachment surface such as a carrier packed bed or even plenary surfacesabove the perforated barrier. Detachment of the cells adhering to theperforated barrier can be performed enzymatically, as is well known inthe art. Such enzymatic treatment can also be combined with flushing theattachment surface with growth medium or a wash buffer and/or withapplying vibrations to the attachment surface.

Reference is now made to FIG. 13 which is a schematic partcross-sectional diagram illustrating a bioreactor system including abioreactor having a perforated barrier and a cell carrier matrix, inaccordance with an embodiment of the bioreactor of the presentapplication. Descriptions of elements presented in FIG. 13 notspecifically detailed herein below, are presented in the description ofFIG. 1 above.

The bioreactor system 1250 is similar to the bioreactor system 50 ofFIG. 1 except that the bioreactor 10 of the bioreactor system 1250further comprises a supporting matrix 1260 which is disposed within thesecond chamber 14B. While the supporting matrix 1260 of the system 1250occupies only a portion of the volume immersed within the growth medium2, in other embodiments of the bioreactor systems, the supporting matrixis configured to extend up to the surface 2A of the growth medium 2 andcan also extend downwards towards the upper surface 12A of theperforated barrier 12. The volume occupied by the support matrix 1260can depend, inter alia, upon the specific application, the resistance ofthe cell supporting matrix 1260 to the flow of the growth medium 2, thefinal amount of required cells or microorganisms and otherconsideration.

According to some embodiments, the bioreactor system 1250 of the presentapplication is configured to allow culturing of adherent cells on anattachment surface such as, for example, a cell carrier matrix packedbed or even plenary surfaces above the perforated barrier. According tosome embodiments, the packed bed of the cell supporting matrix 1260 isconfigured to be positioned above the perforated barrier 12 of thebioreactor 10 allowing grow medium (or other solutions) to circulatethrough the immobile (or less mobile) cell supporting matrix 1260 forfeeding the cells attached to the surface(s) of the cell supportingmatrix 1260.

This arrangement enables constant feeding of the cells attached to thecell supporting matrix 1260, allowing high density cell culturing with ahigh surface to volume ratio and very low sheer forces while constantlyfeeding the cells 3. Such cell supporting matrix 1260 can comprise,inter alia, woven and none woven fibers, electrospin-meshes, plasticbeads, plastic surfaces, biodegradable materials such as, for examplealginate or any other suitable matrices or carriers having twodimensional and/or three dimensional surface(s), as is well known in theart.

According to some embodiments, once there is a need to harvest the cellsattached to the cell supporting matrix 1260, the cells 3 can beenzymatically detached from packed the surface(s) of the cell supportingmatrix 1260 as is well known in the art. The enzymatic treatment can becombined together with flushing the attachment surface with growthmedium or a wash buffer and/or with vibrating of the surface tofacilitate detachment of the adhered cells.

According to some embodiments, Enzymatic detachment of adhered cells canbe performed by adding one or more enzymes to the growth medium 2 andincubation of the adherent cells in the enzyme containing growth mediumfor a prescribed time period. Enzymes useful for performing celldetachment can include but are not limited to a protease (such as, forexample, trypsin, pepsin or papain) or a suitable collagenase, or anycombinations of a collagenase and a protease. Once the cells areharvested from the attachment surface, washing and processing of thecells can be done as described earlier.

Furthermore, in accordance with some embodiments of the bioreactors ofthe present application, the second (upper) chamber of any of thebioreactors disclosed herein is configured to also include a cellsupporting matrix similar to the above disclosed cell supporting matrix1260 which is configured to be introduced into the second chamberthrough any of the openings available in the top part of the bioreactors(such as, for example, through the closable opening 110E of thebioreactor 110 of FIG. 2). While growing non-adherent cells in thebioreactors disclosed herein in which the cells are suspended in thegrowth medium and do not typically adhere to a surface, the bioreactorsdisclosed herein are configured to also be used for growing adherentcells that require some surface or substrate to adhere to. While suchadherent cells can adhere to the perforated barrier of the bioreactor,it can be desirable to increase the surface area available for suchadherent cells in order to increase cell yield. Therefore, in accordancewith some embodiments of the bioreactors of the present application, anyof the bioreactors disclosed herein are configured to include a suitablecell supporting matrix disposed within the second chamber of thebioreactor.

According to some embodiments, the cell supporting matrix can be anytype of cell supporting matrix known in the art to which the cells canadhere. For example, the cell supporting matrix can include a collagenbased matrix, woven and none woven fibers, electro-spin meshes, plastic(polymer based) beads, plastic (polymer based) particles surfaces,biodegradable materials such as, for example alginate, any type ofcollagen or any other suitable matrices or cell carriers having twodimensional and/or three dimensional surface(s) with a high surface tovolume ratio, as is well known in the art.

It is noted that the bioreactors and bioreactor systems disclosed in thepresent application are configured to be used for many differentapplications including, inter alia, the growing of microorganisms likebacteria or any other single cell or multicellular microorganisms,isolated living cells of any type, including but not limited to, livingcells from insects, living cells of invertebrates, living cells ofvertebrates, living mammalian cells, and various different types ofhuman cells. The total volume, shape and other components and/orcharacteristics of the various embodiments of the bioreactors andbioreactor systems disclosed hereinabove are configured to be scaled andadapted to each specific application.

According to some embodiments, the bioreactor 1250 is configured to beused to co-culture together adherent and non-adherent suspended cellsthat need co-culturing were the adherent cells are attached to the cellsupporting matrix 1260 and the suspended non-adhering cells aresuspended in the medium above the perforated bather 12 and below thecell supporting matrix 1260. For example the bioreactor 1250 or anyother of the bioreactors containing a cell supporting matrix areconfigured to be used for culturing of embryonic stem cells which aresuspended non-adherent cells with feeder cells such as adherentfibroblasts.

One example application of the bioreactors and bioreactor systems is thegrowing of cells for cell therapy. Cell therapy is an evolving industrywhere cells are used as therapeutic agents. The cells can be obtainedfrom an autologous source (from the patient) or an allogeneic source(different individual donor). In cases of use of autologous cells, suchas immune-cell therapy (using T cells, and/or B cells and/or dendriticcells, and/or natural killer cells) and/or mesenchymal stem cells. Thetherapeutic dosages can range from several million cells to severalbillion typically cultured in volumes of a few litters (1-20L). Inallogeneic therapies the bio-manufacturing of therapeutic agents canreach volumes of up to thousands of litters per bioreactor.

In some of the embodiments of the bioreactors of the presentapplication, providing for adaptive culturing (using variable mediumlevels) which allow incremental volume changes, media perfusion andrefreshments and high density culturing (such as, but not limited to, inthe bioreactor 20 of FIG. 2) the working volume and bioreactor size canbe advantageously reduced dramatically by about 2-100 fold as comparedto prior art bioreactors. For example, a typical bioreactor having atotal volume in the range of 1-2 litter can be used for culturing thecells required for autologous therapy. Such relatively small bioreactorvolumes can allow the growing of a few billion cells.

According to some embodiments, the ability to use the relatively smallbioreactors of the present application can advantageously save space andreduce operating costs significantly in the facility by allowing the useof many small bioreactors in the same workspace, allowing many smallbioreactors to share common services (such as, for example, by sharing acentral oxygenating supply space, sharing other facilities, such ascomputers, controllers and/or workspace temperature controlling devicesand air conditioning devices and other shareable devices and systems.

It is noted that similar workspace reductions and cost savings can alsobe obtained in larger bioreactors adapted for use in allogeneicculturing in which larger bioreactor volumes are required. Suchallogeneic cell culturing can require using embodiments of thebioreactors disclosed in the present application having bioreactorvolumes in the range of 10-1000 liter (with a typical exemplary, but notobligatory, bioreactor volume of about 100 liter).

It is noted that all the above disclosed bioreactor volume ranges inboth applications of growing allogeneic cells and/or autologous cellsare given by way of example only and are not obligatory. Thus,bioreactors having volumes that are either larger or smaller than theabove ranges can also be used in certain applications and are includedwithin the scope of the volumes of the bioreactors of the presentapplication. For example, in some applications such as, for example,growing algae, bacteria or other microorganisms for obtaining biofuelsor other products, the volume of any of the bioreactors of the presentapplication are configured to be scaled up to volumes much higher than1000 liter.

According to some embodiments, the above mentioned washing methods usingthe above mentioned bioreactors can be applied to any provided cellmass, even if originally incubated in a different bioreactor.

According to some embodiments, the bioreactors' designs as mentionedabove, are configured to allow cell washing and formulating in a verygentle and efficient manner without the need of opening the bioreactorchamber or interfering thereto.

According to some embodiments, the bioreactors' designs as mentionedabove, are configured to allow continuous, optimal and adaptive cellculturing at changing volumes, feeding schemes, activating,manipulating, washing and formulating, all in a closed and automatedbioreactor with minimal sheer force applied onto the cell mass.

It is appreciated that certain features of the bioreactors and systemsthereof disclosed herein, which are, for clarity, described in thecontext of separate embodiments, may also be provided in combination ina single embodiment. Conversely, various features of the bioreactors andsystems thereof disclosed herein, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable sub-combination or as suitable in any other describedembodiment of the bioreactors and systems thereof disclosed herein.Certain features described in the context of various embodiments are notto be considered essential features of those embodiments, unless theembodiment is inoperative without those elements.

Although the bioreactors and systems thereof disclosed herein have beendescribed in conjunction with specific embodiments thereof, it isevident that many alternatives, modifications and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications and variations that fallwithin the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present bioreactors and systems thereofdisclosed herein. To the extent that section headings are used, theyshould not be construed as necessarily limiting.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments may be presented in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope. Accordingly, the descriptionof a range should be considered to have specifically disclosed all thepossible subranges as well as individual numerical values within thatrange. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed subranges such as from 1 to 3,from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., aswell as individual numbers within that range, for example, 1, 2, 3, 4,5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

A skilled artisan would appreciate that the term “medium” may encompassin some embodiments any type of growth medium suitable for growing cells(either eukaryotic or prokaryotic) or any other type of unicellular ormulti-cellular microorganisms. In some embodiments, the term “medium”comprises any type of solution used for cell or microorganism processingincluding but not limited to wash buffers, nutrient buffers, enzymemixtures, selection solutions, and final formulation solutions.

As used herein, in one embodiment the term “about” refers to ±10%. Inanother embodiment, the term “about” refers to ±9%. In anotherembodiment, the term “about” refers to ±9%. In another embodiment, theterm “about” refers to ±8%. In another embodiment, the term “about”refers to ±7%. In another embodiment, the term “about” refers to ±6%. Inanother embodiment, the term “about” refers to ±5%. In anotherembodiment, the term “about” refers to ±4%. In another embodiment, theterm “about” refers to ±3%. In another embodiment, the term “about”refers to ±2%. In another embodiment, the term “about” refers to ±1%.

As used herein, the term “optionally” encompasses the meaning that someelement “is provided in some embodiments and not provided in otherembodiments.” Any particular embodiment disclosed herein may include aplurality of “optional” features unless such features conflict.

Additional objects, advantages, and novel features disclosed herein willbecome apparent to one ordinarily skilled in the art upon examination ofthe following examples, which are not intended to be limiting.Additionally, various embodiments and aspects disclosed herein asdelineated hereinabove and as claimed in the claims section below findsexperimental support in the following examples.

EXAMPLES

The bioreactor system used in the following examples included abioreactor schematically presented in FIG. 14A, which comprises abioreactor similar to that shown in FIG. 1. The perforated barrier wascircular in shape with a 50 cm² diameter and 1 micrometer thickness. Theupper chamber had a conical shape and a 120 cm² top. The total volume ofthe growth chamber (upper chamber) was 250 ml. The term “footprint” usedherein refers to the lower perforated barrier surface area and totalchamber area.

Cells, used to exemplify bioreactor use and effectiveness, wereT-lymphocytes, but this in no way should be considered limiting.

The flow rate used in the Examples was about 2-3 mm per mins. This is arepresentative embodiment of the flow rate for the cells used, whereinthe skilled artisan would appreciate that flow rate may change dependingon cells used. Thus, the flow rate used in the Examples should in no waybe considered limiting. For example, a skilled artisan would appreciatethat when culturing larger cells, such as mesenchymal stem cells (MSC),the flow rate may reach 10 mm per minute, and for even larger cells,such as macrophages, flow rate may reach 20 mm (data not shown).

Example 1: Growth of High Density Cell Cultures

Objective:

High density culturing of cells.

Methods:

Cells (T cell lymphocytes) were grown on a 50 square cm perforatedbarrier system with 150 ml media for 7 days, starting at the maximumknown cell density for these cells of about 4 million cells per ml.Based on knowledge in the art, this is the density at which these cellswould normally be passaged and then maintained at 1 million cells perml. The media was perfused so the total media used was increased but thevolume of media in the chamber remained at 150 ml.

Results:

TABLE 1 Days CM² Cells(E6/ml) Total Cells Cells/cm2 0 50 3.580667,000,000 13,340,000 2 50 5.230 784,500,000 15,690,000 4 50 9.2671,390,050,000 27,801,000 7 50 24.55 3,683,632,500 73,672,650

The data shows that using a bioreactor disclosed herein, the cells weregrown at a density (cells/nil) that is more than 24-fold of the normallyexpected density for these cells (1×10⁶/m1). Similarly, the data showsthat growing cells in a bioreactor system having a footprint of 50 cm²,that starting at 13.3 million per cm² (as opposed to the maximumreported of 10×10⁶/cm²), use of a bioreactor described herein resultedin having 73.6×10⁶ per cm².

Conclusion:

Cells can be grown at high density using a bioreactor comprising a verysmall footprint (50 cm²) of the culturing system. Thus, the bioreactorprovides for a system that allowed optimal and adaptive cell culturingat changing volumes and feeding schemes, allowed for activating,manipulating, feeding, washing, and formulating cells in a closedautomated manner with minimal sheer force (See, Examples 2-3 as well).Additional cell incubators or centrifuges are not required for culturingand collection of cells, respectively.

Example 2: Comparison Cell Cultures: Bioreactors vs. Tissue CultureFlasks

Objective:

Compare culturing cells in a bioreactor comprising a 50 cm2 perforatedbarrier with culturing cells in tissue culture flasks.

Methods:

Cells (T cell lymphocytes) were cultured for 14 days in the same dishesas follows: in either a 50 cm² perforated barrier bioreactor system withperfusion, or a T75 flask without media change, or T75 flask with mediaexchange every 4 days.

Results:

FIGS. 14B-14C present growth curves from two representative culturingexperiments, showing that cells could be continuously grown in abioreactor system having a 50 cm² perforated barrier without the need toreplace media (a pour out/pour in complete exchange), passage or changethe container. Further, that cells grown in the closed continuousbioreactor system (yellow) continued to proliferate for at least 14days, and achieved a total cell number of 1,633,996,000 cells comparedwith only about 4,3200,000 cells in the T75 flask without media change(grey), and only about 300,000,000 cells in the T75 flask with mediachange (blue). The 14-day time frame was used based on the fact thatgrowth of cells in the bioreactor surpassed that in the flasks after aweek. Cells can be cultured for more than two weeks in the bioreactor(data not shown).

Conclusion:

Culturing of cells in a bioreactor system described herein is moreeffective than culturing of cells in flasks even with media exchange.

Example 3: Processing of Cells Grown in a Bioreactor

Objective:

Processing of cells (or microorganisms) includes washing the cells,media replacement, and concentrating the cells. These steps are normallyaccomplished in the prior art by repeated centrifugation and pelletingof the cells. There are two additional technologies known in the art forreplacing media which are a TFF (tangential force filtration)centrifugation and a counter flow centrifuge. The objective of thisexample was to examine cell recovery from a bioreactor as disclosedherein, including the viability of the cells recovered.

Methods:

In the bioreactor system used (demonstrated in FIG. 15A), in order towash the cells and replace the growth media, the wash buffer wasperfused upstream 1510 from the bottom of the bioreactor vessel (lowerchamber 1550), wherein the wash buffer flowed through a first perforatedbarrier 1512 into the upper chamber 1540 and was extracted from thehighest valve 1530. This perfusion flow diluted the media until growthmedia had been replaced by the wash solution. In some embodiments, thevalve 1530 can comprise a perforated barrier or a filter (not shown),configured to prevent the cells from leaving the bioreactor (during theliquids change).

At this point, the final formulation media may be perfused through thesystem, replacing the wash buffer. In addition, in some embodiments,some of the growth media could be drawn-off from the upper chamber(optionally via a second screening perforated barrier (FIG. 15A 1502)configured to prevent the cells from leaving the bioreactor) until alevel where the cells are located, thereby reducing the volume andconcentrating the cells, before the final formulation media is perfused(FIG. 15A). As demonstrated in FIG. 15A, the provided bioreactor with aninverted frustoconical shape allows the cells (or microorganisms)growing mass to float and to elevate to a larger surface, due to thewash solution upstream flow (against gravity direction) and the pressureequilibrium (mass gravity vs. upstream liquid's flow). Further, due toconstant volumetric-flow, a slower flow of the wash solution runsthrough the cells (or microorganisms) mass 3 at the upper and largerareas of the inverted frustoconical shape, which assist in concentratingthe cells mass, and reduces shear forces applied by the wash solutionflow.

In another embodiment, larger volumes of wash solution can be exchangedwith growth media by using a bioreactor with an additional barrierlocated above the level of the cells (when looking at FIG. 15A) andinverting the bioreactor (as shown in FIG. 15B). The bioreactor vesselis configured to be flipped such that the upper chamber (or what is nowthe lower chamber 1540) will have perforated barriers both below 1502and above 1512 the mass of cells. This practically allows more media orwash solution to be downstream perfused due to the larger surface areaof the second barrier (barrier 2 in FIG. 15B). A skilled artisan wouldrecognize that more volume on wider surface area results in the samevelocity (flow rate) so the cells stay near the second barrier (barrier2 in FIG. 15B) and larger volumes of cells mass can be washed.

FIGS. 15C and 15D demonstrate a bioreactor 1590 comprising a vesselconstructed of two frusto-conical parts having same diameter for theirwider base, yet their narrower base can comprise a different diameter.The two parts are sited one on top of the other coaxially joinedtogether at their wider (similar) base. The vessel is divided into threechambers by two perforated barriers; a first perforated barrier 1505 anda second (screening) perforated barrier 1506, which are sealinglydisposed at the walls of the bioreactor's vessel, according to someembodiments. FIG. 15C demonstrates the bioreactor during cell growthstage, where the first lower chamber 1591 (having the narrowest base asits bottom) is configured to be introduced (not shown here) with thegrowth medium, which flows upstream via the first perforated barrier1505, and into the second middle chamber 1592 (which was created by thetwo perforated barrier); the middle chamber is configured to beintroduced with (not shown here) and to accommodate the cells. As shown,the second middle chamber 1592 comprises the area with thelargest/widest cross-section surface 1595, therefore with the slowestmedium's flow rate. According to some embodiments, the aim is not tohave the cells pass this largest/widest area, during the growing stage;this could be achieved for example by controlling the medium's flowvelocity. Above the widest area a second perforated barrier 1506 isshown, which serves as the bottom of upper third chamber 1593, which isconfigured to be introduced with a washing medium (not shown here).

FIG. 15D demonstrates the bioreactor 1590 at its flipped or invertedposition during a washing stage. During the washing stage, the washingmedia is introduced downstream via the third chamber 1593 (not shown)and then down via the cells mass accommodated in the middle chamber 1952and then drained out via the third chamber 1593. The second perforatedbarrier 1506 is configured to prevent cells passage; therefore washedcells are retained in the second middle chamber.

According to some embodiments, a bioreactor configuration such asdemonstrated in FIGS. 15C and 15D, where one base of the vessel is widerthan the other, can serve for growing cells in two steps. In the firststep, the growing can start where the smaller base is facing down, asdemonstrated in FIG. 15C, with very low amounts of cells, allowed togrow to higher surface areas. In the second step when the cell mass isgrown, instead of moving to the cells into a larger chamber of anotherbioreactor, the bioreactor 1590 can be flipped or inverted to have nowthe wider base facing down, as shown in FIG. 15D, allowing the cell masslarger surface area and lower medium's flow rates.

The downstream washing/collecting process was tested in an embodiment ofa bioreactor with a single perforated barrier, wherein three differentsurface velocities were examined near the perforated barrier: 3.6mm/min, 1.8 mm/min, and 1.2 mm/min. Following removal of media with adeep tube (FIG. 15A) 15 ml of cells in growth media remained. The totalwash volume used was 600 ml, wherein the final volume of liquidcomprising the cells was again reduced to 15 ml. Media replacement wasperformed for 40 cycles (Forty (40)×15 ml washes=600 ml total washvolume). There is not a limit to the volume of media that can bereplaced.

Results:

In order to examine the effect of flow rate during exchange of a liquidsolution, the volume of liquid used in the downstream washing/collectionwas maintained but the rate at which the liquid flowed was differed.Thus, an exchange in a shorter time period was a result of a higherflow, and a longer time period was the result of a lower flow rate.

After 30 mins of media exchange at 3.6 mm/min, 60.3% of the cellsrecovered having viability of 87.8%. After 60 mins of media exchange at1.8 mm/min, 100% of the cells were recovered having 91% viability. After90 minutes of media exchange at 1.2 mm/min, 100% of the cells wererecovered with 92.1% viability.

Conclusion:

Media replacement was comparable to other methods known in the art, suchas TFF, which replaces/dilutes 5 volumes. Significantly, using themethod described here to wash and collect cells avoids the high flowrate and shear of the continues flow centrifuge (1-2 liters per minute),as the low flow rates used were 1,000 to 10,000 fold lower with muchless shear.

While certain features of the bioreactors and systems thereof disclosedherein have been illustrated and described herein, many modifications,substitutions, changes, and equivalents will now occur to those ofordinary skill in the art. It is, therefore, to be understood that theappended claims are intended to cover all such modifications and changesas fall within the true spirit of the bioreactors and systems thereofdisclosed herein.

1. A bioreactor for growing cells or microorganisms therein, thebioreactor comprising: a closed vessel enclosing a space therein; afirst barrier having a plurality of pores therein, the first barrier issealingly disposed within the space configured to divide the space intoa first lower chamber and a second upper chamber, wherein the secondchamber is configured to accommodate the growing cells or microorganismstherein, and wherein a diameter of the pores is configured to allow afluid flow solely between the first chamber and the second chamber andvice versa, and wherein the first barrier does not allow cells ormicroorganisms grown in the vessel to pass between the first chamber andthe second chamber; one or more fluid inlet ports for introducing thefluid into the first chamber; and one or more fluid outlet ports forallowing the fluid to exit from the second chamber; and wherein thefluid flow comprises an upstream flow. 2.-4. (canceled)
 5. Thebioreactor according to claim 1, wherein the bioreactor furthercomprises an aligning barrier having a plurality of pores therein; thealigning barrier is sealingly disposed within the space of the firstchamber under the first barrier; the aligning barrier is configured toalign the fluid flow, to control the velocity of the fluid flow, and toprevent bubbles passage.
 6. (canceled)
 7. The bioreactor according toclaim 5, wherein the pores of the aligning barrier comprise conicalshapes.
 8. The bioreactor according to claim 1, wherein the bioreactorfurther comprises an additional screening barrier having a plurality ofpores therein; the screening barrier is disposed within the space of thesecond chamber, at top section of the second chamber, such that thegrowing cells or microorganisms are accommodated between the firstbarrier and the screening barrier; the screening barrier is configuredto prevent the cells passage.
 9. (canceled)
 10. The bioreactor accordingto claim 1, wherein at least the second chamber comprises an increasingtransversal cross sectional area from the bottom to the top of thesecond chamber, configured to provide a fluid velocity gradient in thefluid disposed within the second chamber, such that the velocity of thefluid decreases in a direction from the first barrier towards a topsurface of the fluid.
 11. (canceled)
 12. The bioreactor according toclaim 10, wherein the shape of the transversal cross sections isselected from: a circle, an ellipse, a polygon, and any combinationthereof. 13.-16. (canceled)
 17. The bioreactor according to claim 1,wherein at least one of the following holds true: the one or more fluidoutlet ports comprise a plurality of fluid outlet ports opening atdifferent positions along the height of the second chamber; the firstbarrier is disposed in contact with walls of the vessel; the bioreactorvessel is constructed of at least two parts; the shape of the vessel isselected from: a conical shape, a frustoconical shape, a tapering shape,a cylindrical shape, a polygonal prism shape, a tapering shape having anellipsoidal transversal cross section, a tapering shape having apolygonal transversal cross section, a shape having a cylindrical partand a tapering part and a shape having a conical or tapered part and ahemispherical part, and any combination thereof; at least one of the oneor more fluid outlet ports is configured to be fluidically connected toa pump, which is configured to receive the fluid from the secondchamber, and optionally wherein the pump is further configured torecirculate the fluid back into the first chamber via at least one ofthe fluid inlet ports; the fluid comprises any one of: a growth media, awashing solution, a nutrient solution, a collection solution, aharvesting solution, a storage solution, and any combination thereof;the diameter of each of the pores of the first barrier is selectedbetween about 0.1 to about 40 micrometers; and and any combinationthereof.
 18. (canceled)
 19. The bioreactor according to claim 1, whereinthe first barrier is a fixed non-movable barrier, the barrier isselected from: a flat barrier, a flat barrier inclined at an angle to alongitudinal axis of the bioreactor, a concave barrier with a concaveupper surface facing top of the vessel, a tapering barrier and a conicalbarrier.
 20. The bioreactor according to claim 1, wherein the bioreactorfurther comprises at least one harvesting port disposed in the vicinityof an upper surface of the first barrier configured to harvest cellsfrom the bioreactor.
 21. The bioreactor according to claim 1, whereinthe bioreactor is configured to be inverted.
 22. The bioreactoraccording to claim 1, wherein the bioreactor further comprises asupporting matrix disposed within the second chamber for supporting thecells or microorganisms.
 23. The bioreactor according to claim 1,wherein the bioreactor further comprises a controller, operably coupledand configured to control at least to one of: at least one sensor unitcomprising one or more sensors configured to sense one or more chemicaland/or physical properties of the fluid within the vessel; a pluralityof controllably openable and closable valves configured to control theflow the fluid within the one or more fluid outlet ports outlet andfluid inlet ports; a controllably openable and closable valve configuredto control the flow of fresh liquid fluid from a fluid reservoir into aninlet port of the pump; a heater unit configured to heat the fluidwithin the vessel; a cooling unit configured to cool the fluid withinthe vessel; and a gas valve configured to control the flow of a gascomprising oxygen from an oxygen source into a gas dispersing headdisposed within the vessel.
 24. A method for growing cells ormicroorganisms in a bioreactor, the bioreactor comprising: a closedvessel enclosing a space therein; a first barrier having a plurality ofpores therein, the first barrier is sealingly disposed within the spaceconfigured to divide the space into a first lower chamber and a secondupper chamber, wherein the second chamber is configured to accommodatethe growing cells or microorganisms therein, and wherein a diameter ofthe pores is configured to allow a fluid flow solely between the firstchamber and the second chamber and vice versa, and wherein the firstbarrier does not allow cells or microorganisms grown in the vessel topass between the first chamber and the second chamber; one or more fluidinlet ports for introducing the fluid into the first chamber; and one ormore fluid outlet ports for allowing the fluid to exit from the secondchamber; and wherein the fluid flow comprises an upstream flow; themethod comprises the steps of: introducing cells or microorganisms intothe second chamber of the bioreactor; perfusing the cells ormicroorganisms with the fluid, wherein the perfusing comprisescontrolling the level and/or the rate of flow of the fluid within thebioreactor; growing the cells or microorganism to a desiredconcentration; and harvesting the cells or microorganisms from thebioreactor.
 25. (canceled)
 26. The method according to claim 24, whereinthe step of perfusing further comprises: re-circulating the fluidthrough the first barrier; or oxygenating the fluid; or perfusing thefluid through the first barrier into the second chamber via the firstchamber; or a combination thereof.
 27. The method according to claim 26,wherein the step of re-circulating further comprises at least one of: astep of adding an amount of fresh fluid to the bioreactor; and a step ofdraining an amount of the fluid from the bioreactor.
 28. The methodaccording to claim 24, wherein: the method further comprises step ofincreasing the level of the fluid in the second chamber; or the methodfurther comprises one or more steps of washing the cells ormicroorganisms; or the method further comprises a step of concentratingthe cells by reducing the volume of the fluid within the second chamber;or the method further comprises a step of maintaining the cell mass in afloating position at a specific region in the second chamber, due to abalance between gravity force applied on the cell mass and selectedvelocity of the upstream fluid flow; or the method further comprises astep of co-culturing the cells with additional different cells; or themethod further comprises a step of controlling the concentration ofgaseous materials in the headspace of the second chamber, wherein thegaseous materials comprise oxygen and CO₂; or the method furthercomprises a step controlling at least one of: dissolved oxygen level andpH level of the fluid, via the steps of perfusing and/or adjusting; orthe method further comprises a step of adding cytokines to the culturemedia or co-culturing the cells with cytokine secreting cells attachingcytokine; or the method further comprises a step of coating the barrierwith an antibody; or any combination thereof.
 29. The method accordingto claim 24, wherein the cells are adherent cells and the method furthercomprises a step of allowing the cells to attach to one or more surfacesdisposed within the second chamber.
 30. The method according to claim29, wherein the one or more surfaces are selected from the groupconsisting of: the upper surface of the first barrier, the surface ofthe walls of the second chamber, the surface of a cell supporting matrixdisposed within the second chamber, and any combination thereof. 31.(canceled)
 32. The method according to claim 28, wherein: the cells areT-cells and the additional different cells are cytokine secreting cells;or the cells are T-cells and the additional different cells are antigenpresenting cells; or the cells are embryonic stem cells and theadditional different cells are feeder cells.
 33. The method according toclaim 24, wherein the steps of introducing, perfusing, controlling,growing, washing, and harvesting the cells are continuous and performedin or from the second chamber.